U.S. patent number 7,888,350 [Application Number 12/294,599] was granted by the patent office on 2011-02-15 for 3,7-diamino-10h-phenothiazine salts and their use.
This patent grant is currently assigned to Wista Laboratories Ltd.. Invention is credited to Thomas Craven Baddeley, Charles Robert Harrington, David Horsley, Colin Marshall, Janet Elizabeth Rickard, James Peter Sinclair, John Mervyn David Storey, Claude Michel Wischik.
United States Patent |
7,888,350 |
Wischik , et al. |
February 15, 2011 |
**Please see images for:
( Reexamination Certificate ) ** |
3,7-diamino-10H-phenothiazine salts and their use
Abstract
This invention pertains generally to the field of phenothiazine
compounds, and more particularly to certain stably reduced
phenothiazine compounds, specifically, certain 3,7
diamino-10H-phenothiazine (DAPTZ) compounds of the following
formula wherein: each of R.sup.1 and R.sup.9 is independently
selected from: --H; C.sub.1-4alkyl; C.sub.2-4alkenyl; and
halogenated C.sub.1-4alkyl; each of R.sup.3NA and R.sup.3NB is
independently selected from: --H; C.sub.1-4alkyl; C.sub.2-4alkenyl;
and halogenated C.sub.1-4alkyl; each of R.sup.7NA and R.sup.7NB is
independently selected from: --H; C.sub.1-4alkyl; C.sub.2-4alkenyl;
and halogenated C.sub.1-4alkyl; each of HX.sup.1 and HX.sup.2 is
independently a protic acid; and pharmaceutically acceptable salts,
solvates, and hydrates thereof. These compounds are useful as
drugs, for example, in the treatment of tauopathies, such as
Alzheimer's disease, and also as prodrugs for the corresponding
oxidized thioninium drugs (for example, methythioninium chloride,
MTC).
Inventors: |
Wischik; Claude Michel
(Aberdeen, GB), Rickard; Janet Elizabeth (Aberdeen,
GB), Harrington; Charles Robert (Aberdeen,
GB), Horsley; David (Aberdeen, GB), Storey;
John Mervyn David (Old Aberdeen, GB), Marshall;
Colin (Old Aberdeen, GB), Sinclair; James Peter
(Old Aberdeen, GB), Baddeley; Thomas Craven (Old
Aberdeen, GB) |
Assignee: |
Wista Laboratories Ltd.
(Singapore, SG)
|
Family
ID: |
38421429 |
Appl.
No.: |
12/294,599 |
Filed: |
March 28, 2007 |
PCT
Filed: |
March 28, 2007 |
PCT No.: |
PCT/GB2007/001103 |
371(c)(1),(2),(4) Date: |
September 25, 2008 |
PCT
Pub. No.: |
WO2007/110627 |
PCT
Pub. Date: |
October 04, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090054419 A1 |
Feb 26, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60786690 |
Mar 29, 2006 |
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Current U.S.
Class: |
514/226.2;
544/37 |
Current CPC
Class: |
C07D
279/20 (20130101); A61P 25/28 (20180101); A61P
7/06 (20180101); A61P 1/16 (20180101); A61P
25/20 (20180101); A61P 9/00 (20180101); A61P
33/06 (20180101); A61K 31/5415 (20130101); A61P
3/10 (20180101); A61P 33/02 (20180101); A61P
25/00 (20180101); A61P 13/12 (20180101); A61P
43/00 (20180101); A61P 13/08 (20180101); A61P
33/00 (20180101); A61P 29/00 (20180101); A61P
31/12 (20180101); A61P 25/14 (20180101); A61P
35/00 (20180101); A61P 25/16 (20180101); A61P
31/04 (20180101); A61P 31/14 (20180101); C07D
279/22 (20130101); C09B 21/00 (20130101); A61P
31/18 (20180101); Y02A 50/393 (20180101); Y02A
50/411 (20180101); Y02A 50/30 (20180101) |
Current International
Class: |
C07D
279/18 (20060101); A61K 31/5415 (20060101) |
Field of
Search: |
;544/37 ;514/226.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
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|
|
4403091 |
|
Aug 1994 |
|
DE |
|
0 457 295 |
|
Nov 1991 |
|
EP |
|
0 618 968 |
|
Oct 1994 |
|
EP |
|
0 737 671 |
|
Oct 1996 |
|
EP |
|
0 909 814 |
|
Apr 1999 |
|
EP |
|
0 911 390 |
|
Apr 1999 |
|
EP |
|
1067386 |
|
Oct 2001 |
|
EP |
|
2 788 436 |
|
Jul 2000 |
|
FR |
|
6-289015 |
|
Oct 1994 |
|
JP |
|
WO 89/03993 |
|
May 1989 |
|
WO |
|
WO 93/03177 |
|
Feb 1993 |
|
WO |
|
WO 93/03369 |
|
Feb 1993 |
|
WO |
|
WO 93/11231 |
|
Jun 1993 |
|
WO |
|
WO 95/05601 |
|
Aug 1994 |
|
WO |
|
WO 95/05466 |
|
Feb 1995 |
|
WO |
|
WO 96/04915 |
|
Feb 1996 |
|
WO |
|
WO 96/05837 |
|
Feb 1996 |
|
WO |
|
WO 96/30766 |
|
Oct 1996 |
|
WO |
|
WO 99/62548 |
|
Dec 1999 |
|
WO |
|
WO 01/53340 |
|
Jul 2001 |
|
WO |
|
WO 02/03972 |
|
Jan 2002 |
|
WO |
|
WO 02/04025 |
|
Jan 2002 |
|
WO |
|
WO 02/055720 |
|
Jul 2002 |
|
WO |
|
WO 02/059150 |
|
Aug 2002 |
|
WO |
|
WO 02/075318 |
|
Sep 2002 |
|
WO |
|
WO 03/007933 |
|
Jan 2003 |
|
WO |
|
WO 2005/030676 |
|
Apr 2005 |
|
WO |
|
WO 2006/032879 |
|
Mar 2006 |
|
WO |
|
WO 2007/110629 |
|
Oct 2007 |
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WO |
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Other References
Vippagunta et al, "Crystalline Solids" Advanced Drug Delivery
Reviews, vol. 48, pp. 3-26 (2001). cited by examiner .
Gavezzotti, "Are Crystal Structures Predictable?" Accounts of
Chemical Research, vol. 27, pp. 309-314 (1994). cited by examiner
.
Cecil Textbook of Medicine (20th Edition, vol. 2, 1996, pp.
1739-1747). cited by examiner .
International Search Report for PCT/GB2007/001103 dated Aug. 30,
2007 (4 pgs.). cited by other .
Zhang, et al., "Methylene Blue Prevents Neurodegeneration Caused by
Rotenone in the Retina", Neurotoxicity Research, vol. 9, No. 1,
2006, pp. 47-57. cited by other .
U.S. Appl. No. 60/960,544, filed Oct. 3, 2007, Wischik, et al.
cited by other .
U.S. Appl. No. 60/996,177, filed Nov. 5, 2007, Wischik, et al.
cited by other .
U.S. Appl. No. 61/077,281, filed Jul. 1, 2008, Wischik, et al.
cited by other .
U.S. Appl. No. 60/945,006, Jun. 19, 2007, Wischik, et al. cited by
other .
Aizawa et al., "Microtubule-binding domain of tau proteins,"
Journal of Biological Chemistry, 1988, vol. 263, pp. 7703-7707.
cited by other .
Anderton et al., "Dendritic Changes in Alzheimer's Disease and
Factors That May Underlie these Changes," Prog. Neurobiol., Aug.
1998, pp. 595-609, vol. 55, No. 6. cited by other .
Avila et al., Assorted Proteins, Harwood Aend Publishers,
Amsterdam, 1997. cited by other .
Bancher et al., "Accumulation of abnormally phosphorylated .tau.
precedes the formation of neurofibrillary tangles in Alzheimer's
disease," Brain Research, 1989, vol. 477, pp. 90-99. cited by other
.
Biernat et al., "The switch of tau protein to an Alzheimer-like
state includes the phosphorylation of two serine-proline motifs
upstream of the microtubule binding region," EMBO Journal 11, 1992,
pp. 1593-1597. cited by other .
Braak et al., "Alzheimer's Disease: Transiently Developing
Dendritic Changes in Pyramidal Cells of Sector CA1 of the Ammon's
Horn," Acta Neuropathol., 1997, pp. 323-325, vol. 93. cited by
other .
Brandt R., "Cytoskeletal Mechanisms of Axon Outgrowth and
Pathfinding," Cell Tissue Res., 1998, pp. 181-189, vol. 292. cited
by other .
Brion et al., "Characterization of a Partial cDNA Specific for the
High Molecular Weight Microtubule-Associated Protein MAP2 That
Encodes Epitopes Shared with Paired Helical Filaments of
Alzheimer's Disease," Dementia, 1990, pp. 304-315, vol. 1. cited by
other .
Callaway et al, "Methylene blue restores spatial memory retention
impaired by an inhibitor of cytochrome oxidase in rats",
Neuroscience Letters, 332, (2002), pp. 83-86. cited by other .
Caputo et al., "Amyloid-like properties of a synthetic peptide
corresponding to the carboxy terminus of (.beta.-amyloid protein
precursor," Archives of Biochemistry and Biophysics, 1992, vol.
292, pp. 199-205. cited by other .
Condamines et al., "New immunoassay for the mapping of
neurofibrillary degeneration in Alzheimer's disease using two
monoclonal antibodies against human paired helical filament tau
proteins," Neuroscience Letters, Jun. 9, 1995, vol. 192, No. 2, pp.
81-84. cited by other .
Cudd et al., "Pharmacokinetics and toxicity of tolonium chloride in
sheet", Vet Human Toxicol, 38(5), Oct. 1996, pp. 329-334. cited by
other .
Day R., "How to Write and Publish a Scientific Paper," 1983, pp.
124-127, ISI Press, Philadelphia, PA. cited by other .
De Ancos et al., Journal of Biological Chemistry, 1993, pp.
7976-7982, vol. 268(11). cited by other .
DeTure et al., "In vitro assembly of Alzheimer-like filaments. How
a small cluster of charged residues in tau and MAP2 controls
filament morphology," Journal of Biological Chemistry, 2002, vol.
277, pp. 34755-34759. cited by other .
Epstein, J.B. et al, "The utility of toluidine blue application as
a diagnostic aid in patients previously treated for upper
oropharyngeal carcinoma", Oral medicine, (1997), pp. 537-547, vol.
83, No. 5. cited by other .
Fasulo et al., "Overexpression of Alzheimer's PHF core tau
fragments: implications for the tau truncation hypothesis," Rapid
Science Publishers, Alzheimer's Research, vol. 2, No. 5, pp.
195-200, Oct. 1996. cited by other .
Friedhoff et al., Biochemistry, 1998, pp. 10223-10230, vol. 37.
cited by other .
Friedhoff et al., PNAS, 1998, pp. 15712-15717, vol. 95. cited by
other .
Garcini et al., "In Vitro Conditions for the Self-Polymerization of
the Microtubule-Associated Protein, Tau Factor," J. Biochem., 1987,
pp. 1415-1421, vol. 102, No. 6. cited by other .
Garcini et al., Self Assembly of Microtubule Associated Protein TAU
into Filaments Resembling those found in Alzheimer Disease,
Biochemical and Biophysical Research Communications, 1988, pp.
790-797. cited by other .
Garcini et al., "Tau Factor Polymers are Similar to Paired Helical
Filaments of Alzheimer's Disease," 1988, pp. 150-154, Elsevier
Science Publishers B.V. cited by other .
Giannetti et al., "Fibers of tau fragments, but not full length
tau, exhibit a cross .beta.-structure: implications for the
formation of paired helical filaments," Protain Science, 2000, vol.
9, pp. 2427-2435. cited by other .
Goedert et al., "Tau Proteins of Alzheimer Paired Helical
Filaments: Abnormal Phosphorylation of All Six Brain Isoforms,"
Neuron, Jan. 1992, pp. 159-168, vol. 8. cited by other .
Goedert M. et al., "Cloning and sequencing of the cDNA encoding a
core protein of the paired helical filament of Alzheimer disease:
Identification as the microtubule-associated protein tau," Proc.
Natl. Acad. Sci. USA, Jun. 1988, vol. 85, pp. 4051-4055. cited by
other .
Gotz et al., "Tau filaments formation in transgenic mice expressing
P301L tau," J. Biol. Chem., Jan. 5, 2001, vol. 276(1), pp. 529-534.
cited by other .
Grover et al., "5' Splice Site Mutations in Tau Associated with the
Inherited Dementia FTDP-17 Affect a Stem-Loop Structure That
Regulates Alternative Splicing of Exon 10*," The Journal of
Biological Chemistry, May 21, 1999 Issue, pp. 15134-15143, vol.
274, No. 21. cited by other .
Grundke-Iqbal et al., "Abnormal phosphorylation of
microtubule-associated protein T (tau) in Alzheimer cytoskeletal
pathology," Proc. Natl. Acad. Sci. USA, 1986, vol. 83, pp.
4913-4917. cited by other .
Hagestedt et al., "Tau protein becomes long and stiff upon
phosphorylation: correlation between paracrystalline structure and
degree of phosphorylation," The Journal of cell biology, 1989, vol.
109, pp. 1643-1651. cited by other .
Harrington et al., "Competitive Elisa for the Measurement of Tau
Protein in Alzheimer's Disease," Journal of Immunological Methods,
1990, pp. 261-271, vol. 134. cited by other .
Harrington et al., "Measurement of Distinct Immunochemical
Presentations of Tau Protein in Alzheimer Disease," Proc. Natl.
Acad. Sci., Jul. 1991, pp. 5842-5846, vol. 88. cited by other .
Holoubek et al., "Toluidine blue in bleeding associated with
thrombopenia", J.A.M.A., Jan. 22, 1949, vol. 139, No. 4, pp.
214-216. cited by other .
Hutton et al., "Association of Missense and 5'-splice-site
Mutations in Tau With the Inherited Dementia FTDP-17," Nature, Jun.
18, 1998, pp. 702-705, vol. 393. cited by other .
Ishiguro et al., "A novel tubulin-dependent protein kinase forming
a paired helical filament epitope on tau," J. Biochem, 1988, vol.
104, pp. 319-321. cited by other .
Ishiguro et al., "A serine/threonine proline kinase activity is
included in the tau protein kinase fraction forming a paired
helical filament epitope," Neuroscience Letters, 1991, vol. 128,
pp. 195-198. cited by other .
Ishiguro et al., "Phosphorylation sites on tau by tau protein
kinase I, a bovine derived kinase generating an epitope of paired
helical filaments," Neuroscience Letters, 1992, vol. 148, pp.
202-206. cited by other .
Ishiguro et al., "Tau protein kinase I converts normal tau protein
into A68-like component of paired helical filaments," Journal of
Biological Chemistry, 1992, vol. 267, pp. 10897-10901. cited by
other .
Ishihara et al., "Age-Dependent Emergence and Progression of a
Tauopathy in Transgenic Mice Overexpressing the Shortest Human Tau
Isoform," Neuron, Nov. 1999, pp. 751-762, vol. 24. cited by other
.
Ito, A. et al., "Enhancing effect of ascorbate on toluidine
blue-photosensitization of yeast cells" Photochemistry and
Photobiology, (1982), pp. 501-505, vol. 35. cited by other .
Jakes et al., "Identification of 3- and 4-repeat Tau Isoforms
within the PHF in Alzheimer's Disease," The EMBO Journal, 1991, pp.
2725-2729, vol. 10, No. 10. cited by other .
Janciauskiene et al., "In vitro amyloid fibril formulation from
.alpha.1-antitrypsin," Bio Chem, 1995, vol. 375, pp. 103-109. cited
by other .
Kaech et al., "Cytoskeletal Plasticity in Cells Expressing Neuronal
Microtubule-Associated Proteins," Neuron, Dec. 1996, pp. 1189-1199,
vol. 17. cited by other .
Kiese et al., "Comparative studies on the effects of toluidine blue
and methylene blue on the reduction of ferrihaemoglobin in man and
dog" , Europ. J. Clin. Pharmacol., 1972, vol. 4, pp. 115-118. cited
by other .
Ksiezak-Reding et al., "Mass and Physical Dimensions of Two
Distinct Populations of Paired Helical Filaments," Neurobiology of
Aging, 1993, pp. 11-18, vol. 15, No. 1. cited by other .
Ksiezak-Reding et al., "Structural Stability of Paired Helical
Filaments Requires Microtubule-Binding Domains of Tau: A Model for
Self-Association," Neuron, 1991, pp. 717-728, vol. 6. cited by
other .
Lai et al., "Examination of Phosphorylated Tau Protein as a
PHF-Precursor at Early State Alzheimer's Disease," Neurobiology of
Aging, 1995, pp. 433-445, vol. 16, No. 3. cited by other .
Ledesma et al., "Implication of brain cdc2 and MAP2 kinases in the
phosphorylation of tau protein in Alzheimer's disease," FEBS, 1992,
vol. 308, No. 2, pp. 218-224. cited by other .
Lee et al., untitled, Science, 1992., vol. 251. cited by other
.
Lee et al., "A68: A Major Subunit of Paired Helical Filaments and
Derivatized Forms of Normal Tau," Science, 1991, pp. 675-678, vol.
251. cited by other .
Martinez et al., "Methylene blue alterse retention of inhibitory
avoidance responses", Physiol. Psychol., 1978, vol. 6(63), pp.
387-390. cited by other .
Masuda, M., et al., Small molecule inhibitors of .alpha.-synuclein
filament assembly. Biochemistry, (2006), pp. 6085-6094, 45. cited
by other .
May et al., "Reduction and uptake of methylene blue by human
erythrocytes", Am J Physiol Cell Physiol , 286, 2004, pp.
C390-C1398. cited by other .
Mena et al., "A Progressive Depsotion of Paired Helical Filaments
(PHF) in the Brain Characterizes the Evolution of Dementia in
Alzheimer's Disease," Journal of Neuropathology and Experimental
Neurology, 1991, pp. 474-490. cited by other .
Mena et al., "Monitoring Pathological Assembly of tau and
.beta.-Amyloid Proteins in Alzheimer's Disease," Acta Neuropathol.,
1994, pp. 50-56. cited by other .
Mena et al., "Staging the Pathological Assembly of Truncated tau
Protein into Paired Helical Filaments in Alzheimer's Disease," Acta
Neuropathol, 1995, pp. 633-641. cited by other .
Muller T., "Light-microscopic demonstration of methylene blue
accumulation sites in mouse brain after supravital staining", Acta
Anat., 1992, vol. 144, pp. 39-44. cited by other .
Murphy et al., "Cyclic-voltametric studies of some phenothiazine
dyes," J. Chem. Soc., Faraday Trans., 1984, vol. 80, pp. 2745-2750.
cited by other .
Novak et al., "Molecular Characterization of the Minimal Protease
Resistant Tau Unit of the Alzheimer's Disease Paired Helical
Filament," The EMBO Journal, 1993, pp. 365-370, vol. 12, No. 1.
cited by other .
Pedrotti et al., Biochemistry, 1994, pp. 8798-8806, vol. 33. cited
by other .
Perez et al., "In vitro assembly of tau protein: Mapping the
regions involved in filament formation," Biochemistry, 2001, vol.
40, 5983-5991. cited by other .
Perez-Tur et al., "Neurodegenerative disease of Guam: Analysis of
TAU," American Academy of Neurology, 1999, vol. 53, pp. 411-412.
cited by other .
Pickhardt et al., "Anthraquinones inhibit tau aggregation and
dissolve Alzheimer paired helical filaments in vitro and in cells,"
Journal of Biological Chemistry, 2005, vol. 280, pp. 3628-3635.
cited by other .
Poulter et al., "Locations and immunoreactivities of
phosphorylation sites on bovine and porcine tau proteins and a
PHF-tau fragment," The Journal of Biological Chemistry, 1993, vol.
268, No. 13, pp. 9636-9644. cited by other .
Rumbolz et al., "Use of protamine sulfate and toluidine blue for
abnormal uterine bleeding," Am. J. Obst. & Gynec., May 1952,
vol. 63, No. 5, pp. 1029-1037. cited by other .
Sato-Harada et al., "Microtubule-associated Proteins Regulate
Microtubule Function as the Track for Intracellular Membrane
Organelle Transports," Cell Structure and Function, 1996, pp.
283-295, vol. 21. cited by other .
Schneider et al., "Phosphorylation that detaches tau protein from
microtubules (Ser262, Ser214) also protects it against aggregation
into Alzheimer paired helical filaments," Biochemistry, 1999, vol.
38, pp. 3549-3558. cited by other .
Shojania, A.M. et al., "The effect of toluidine blue and methylene
blue in immunochemical reactions in vitro", Clinical Immunology and
Immunopathology, (1987), pp. 223-228, 43. cited by other .
Smith et al., "The molecular pathology of Alzheimer's disease: are
we any closer to understanding the neurodegenerative process?,"
Neuropathology and Applied Neurobiology, 1994, pp. 322 338, vol.
20, XP002002176. cited by other .
Taniguchi et al, "Inhibition of heparin-induced tau filament
formation by phenothiazines, polyphenols, and porphyrins." JBC
Papers in Press, 2004, Manuscript M408714200. cited by other .
Tint et al., "Acute Inactivation of Tau Has No Effect on Dynamics
of Microtubules in Growing Axons of Cultured Sympathetic Neurons,"
The Journal of Neuroscience, Nov. 1, 1998, pp. 8661-8673, vol. 18,
No. 21. cited by other .
Van Rossum et al., "Cytoskeletal Dynamics in Dendritic Spines:
Direct Modulation by Glutamate Receptors?," Trends Neurosci., 1992,
pp. 290-295, vol. 22. cited by other .
Varani et al., "Structure of tau exon 10 splicing regulatory
element RNA and destabilization by mutations of frontotemporal
dementia and parkinsonism linked to chromosome 17," Proc. Natl.
Acad. Sci. USA, 1999, vol. 96, pp. 8229-8234. cited by other .
Von Bergen et al., "Assembly of tau protein into Alzheimer's paired
helical filaments depends on a local sequence motif forming beta
structure," Proceedings of the National Academy of Sciences of USA,
National Academy of Science, May 9, 2000, vol. 97, No. 10, pp.
5129-5134. cited by other .
Wille et al., Alzheimer-like paired helical filaments and
antiparallel dimars formed from microtubule-associated protein tau
in vitro, J. Cell Biol., 1992, pp. 573-584, vol. 118. cited by
other .
Wischik C., "Molecular Neuropathology of Alzheimer's Disease,"
1989, pp. 44-70. cited by other .
Wischik C., "Molecular neuropathology of Alzheimer's disease," John
Libbey & Co., 1991, pp. 239-250. cited by other .
Wischik et al. "Quantitative Analysis of Tau Protein in Paired
Helical Filament Preparations: Implications for the Role of Tau
Protein Phosphorylation in PHE Assembly in Alzheimer's Disease,"
Neurobiology of Aging, 1995, pp. 409-431, vol. 16, No. 3. cited by
other .
Wischik et al., "Author's Response to Commentaries," pp. 423-431.
cited by other .
Wischik et al., "Isolation of a Fragment of Tau Derived From the
Core of the Paired Helical Filament of Alzheimer Disease," Proc.
Natl. Acad. Sol. USA, Jun. 1998, pp. 4506-4510, vol. 85. cited by
other .
Wischik et al., "Modelling Prior-like Processing of Tau Protein in
Alzheimer's disease for Pharmaceutical Development," Harwood Acad.
Publishers, 1997, pp. 185-241. cited by other .
Wischik et al., "Selective inhibition of Alzheimer disease-like tau
aggregation by phenothiazines," Proc. Natl. Acad. Sci. USA, 1996,
pp. 11213-11218, vol. 93. cited by other .
Wischik et al., "Structural Characterization of the Core of the
Paired Helical Filament of Alzheimer Disease," Proc. Natl. Acad.
Sci. USA, Jul. 1998, pp. 4884 4888, vol. 85. cited by other .
Wischik et al., "Structure, Biochemistry and Molecular Pathogenesis
of Paired Helical Filaments in Alzheimer's Disease," Pathobiology
of Alzheimer's Disease, 1995, pp. 10-39. cited by other .
Wischik et al., "Subunit Structure of Paired Helical Filaments in
Alzheimer's Disease," The Journal of Cell Biology, 1985, p.
1905-1913. cited by other .
Wischik et al., "Quantitative Analysis of Tau Protein in Paired
Helical Filament Preparations: Implications for the Role of Tau
Protein Phosphorylation in PHF Assembly in Alzheimer's Disease,"
Neurobiology of Aging, 1995, pp. 409-431, vol. 16, No. 3. cited by
other .
Wischik, "Cell biology of the Alzheimer tangle," Current Opinion in
Cell Biology, 1989, vol. 1, pp. 115-122. cited by other .
Yen et al., "Alzheimer's Neurofibrillary Tangles Contain Unique
Epitopes and Epitopes in Common With the Heat-Stable Microtubule
Associated Proteins Tau and MAP.sub.2," AJP, Jan. 1987, vol. 126.
cited by other .
Caputo et al., "The amyloid proteins of Alzheimer's disease as
potential targets for drug therapy," Neurobiology of Aging, vol.
10, 1989, pp. 451-461. cited by other .
Harada et al., "Altered Microtubule Organization in Small-Calibre
Axons of Mice Lacking Tau Protein," Letters to Nature, vol. 369,
1994, pp. 488-489. cited by other .
Kohler & Co., "Toluidinblau," Drug Information, Alsbach,
Germany, Jul. 1997. cited by other .
Lai R., "The Role of Abnormal Phosphorylation of Tau Protein in the
Development of Neurofibrillary Pathology in Alzheimer's
Disease"-Submitted for the Degree of Doctor of Philosophy at the
University of Cambridge, 1994, pp. 1-243. cited by other .
Lee et al., "Tau Proteins and their significance in the
Pathobiology of Alzheimer's Disease," Pathobiology of Alzheimer's
Disease, 1995, pp. 41-58. cited by other .
Mashberg A., "Tolonium (Toluidine blue) rinse - a screening method
for recognition of squanous carcinoma - continuing study of
oral-cancer 4," Jama-Journal of the American Medical Association,
vol. 245, No. 23, 1981, pp. 2408-2410. cited by other .
Wischik C.M., Thesis "The Structure and Biochemistry of Paired
Helical Filaments in Alzheimer's Disease," Part I and II, May 1989.
cited by other .
Wischik et al., "The molecular basis of tau protein pathology in
Alzheimer's disease and related neurodegenerative dementias," In
Neurobiology of Alzheimer's Disease (Eds. D. Dawbarn & S.J.
Allen) Oxford University Press, Oxford, 2001, pp. 103-206. cited by
other.
|
Primary Examiner: Habte; Kahsay T
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Phase of International
Application No. PCT/GB2007/001103, filed Mar. 28, 2007, which was
published in English on Oct. 3, 2007 as WO 2007/110627; and claims
benefit of the filing date of U.S. patent application No.
60/786,690 filed 29 Mar. 2006; the contents of which are
incorporated herein by reference in their entirety.
Claims
The invention claimed is:
1. A compound selected from compounds of the following formula and
pharmaceutically acceptable salts thereof: ##STR00057## wherein:
each of R.sup.1 and R.sup.9 is independently selected from: --H,
C.sub.1-4alkyl, C.sub.2-4-alkenyl, and halogenated C.sub.1-4alkyl;
each of R.sup.3NA and R.sup.3NB is independently selected from:
--H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl; each of R.sup.7NA and R.sup.7NB is independently
selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl; each of HX.sup.1 and HX.sup.2 is
independently a protic acid.
2. A compound according to claim 1, wherein each of R.sup.1 and
R.sup.9 is independently --H, -Me, -Et, or --CF.sub.3.
3. A compound according to claim 1, wherein each of R.sup.1 and
R.sup.9 is independently --H, -Me, or -Et.
4. A compound according to claim 1, wherein R.sup.1 and R.sup.9 are
the same.
5. A compound according to claim 1, wherein each of R.sup.1 and
R.sup.9 is independently --H.
6. A compound according to claim 1, wherein each of R.sup.3NA and
R.sup.3NB independently -Me, -Et, -nPr, -nBu,
--CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3.
7. A compound according to claim 1, wherein R.sup.3NA and R.sup.3NB
are the same.
8. A compound according to claim 1, wherein each of R.sup.7NA and
R.sup.7NB is independently -Me, -Et, -nPr, -nBu,
--CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3.
9. A compound according to claim 1, wherein R.sup.7NA and R.sup.7NB
are the same.
10. A compound according to claim 1, with the proviso that: at
least one of R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB is
other than -Et.
11. A compound according to claim 1, with the proviso that: if:
each of R.sup.1 and R.sup.9 is --H; then: R.sup.3NA and R.sup.3NB
and R.sup.7NA and R.sup.7NB are not each -Et.
12. A compound according to claim 1, wherein each of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is
independently selected from: --NMe.sub.2, --NEt.sub.2,
--N(nPr).sub.2, --N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
13. A compound according to claim 1, wherein the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) are the
same, and are selected from: --NMe.sub.2 and --NEt.sub.2.
14. A compound according to claim 1, wherein the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) are the
same.
15. A compound according to claim 1, with the proviso that: each of
the groups --N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB)
is other than --NEt.sub.2.
16. A compound according to claim 1, wherein the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) are the
same, and are selected from: --NMe.sub.2, --N(nPr).sub.2,
--N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
17. A compound according to claim 1, wherein each of the groups
--N(R.sup.3NA)(R.sup.3NB).sub.and --N(R.sup.7NA)(R.sup.7NB) is:
--NMe.sub.2.
18. A compound according to claim 1, wherein each of HX.sup.1 and
HX.sup.2 is independently a monoprotic acid.
19. A compound according to claim 1, wherein each of HX.sup.1 and
HX.sup.2 is independently a hydrohalide acid.
20. A compound according to claim 1, wherein each of HX.sup.1 and
HX.sup.2 is independently selected from HCl, HBr, and Hl.
21. A compound according to claim 1, wherein HX.sup.1 and HX.sup.2
are each HCl.
22. A compound according to claim 1, wherein HX.sup.1 and HX.sup.2
are each HBr.
23. A compound according to claim 1, selected from compounds of the
following formula, and pharmaceutically acceptable salts thereof:
##STR00058##
24. A compound according to claim 1, selected from compounds of the
following formula, and pharmaceutically acceptable salts thereof:
##STR00059##
25. A compound according to claim 1, in substantially purified
form.
26. A compound according to claim 1, wherein one or more of the
carbon atoms of the compound is .sup.11C.
27. A pharmaceutical composition comprising a compound according to
claim 1, and a pharmaceutically acceptable carrier or diluent.
28. A method of preparing a pharmaceutical composition comprising
admixing a compound according to claim 1, and a pharmaceutically
acceptable carrier or diluent.
29. A pharmaceutical composition comprising a compound of the
following formula: ##STR00060## and a pharmaceutically acceptable
carrier or diluent.
30. A pharmaceutical composition comprising a compound of the
following formula: ##STR00061## and a pharmaceutically acceptable
carrier or diluent.
Description
TECHNICAL FIELD
This invention pertains generally to the field of phenothiazine
compounds, and more particularly to certain stably reduced
phenothiazine compounds, specifically, certain
3,7-diamino-10H-phenothiazine (DAPTZ) compounds, for example,
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride) and N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine
bis(hydrogen iodide). These compounds are useful as drugs, for
example, in the treatment of tauopathies, such as Alzheimer's
disease, and also as prodrugs for the corresponding oxidized
thioninium drugs (for example, methythioninium chloride, MTC).
BACKGROUND
A number of patents and publications are cited herein in order to
more fully describe and disclose the invention and the state of the
art to which the invention pertains. Each of these references is
incorporated herein by reference in its entirety into the present
disclosure, to the same extent as if each individual reference was
specifically and individually indicated to be incorporated by
reference.
Throughout this specification, including the claims which follow,
unless the context requires otherwise, the word "comprise," and
variations such as "comprises" and "comprising," will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from "about" one particular
value, and/or to "about" another particular value. When such a
range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by the use of the
antecedent "about," it will be understood that the particular value
forms another embodiment.
Conditions of dementia are frequently characterised by a
progressive accumulation of intracellular and/or extracellular
deposits of proteinaceous structures such as .beta.-amyloid plaques
and neurofibrillary tangles (NFTs) in the brains of affected
patients. The appearance of these lesions largely correlates with
pathological neurofibrillary degeneration and brain atrophy, as
well as with cognitive impairment (see, e.g., Mukaetova-Ladinska,
E. B. et al., 2000, Am. J. Pathol., Vol. 157, No. 2, pp. 623-636).
Methythioninium chloride (MTC) and other diaminophenothiazines have
been described as inhibitors of protein aggregation in such
diseases, that is, diseases in which proteins aggregate
pathologically (see, for example, WO 96/30766 and WO
02/055720).
Methythioninium chloride (MTC) is currently used to treat
methemoglobinemia (a condition that occurs when the blood cannot
deliver oxygen where it is needed in the body). MTC is also used as
a medical dye (for example, to stain certain parts of the body
before or during surgery); a diagnostic (for example, as an
indicator dye to detect certain compounds present in urine); a mild
urinary antiseptic; a stimulant to mucous surfaces; a treatment and
preventative for kidney stones; and in the diagnosis and treatment
of melanoma.
MTC has been used to treat malaria either singly (see, e.g.,
Guttmann, P. and Ehrlich, P., 1891, "Uber die wirkung des
methylenblau bei malaria," Berl. Kiin. Woschenr., Vol. 28, pp.
953-956) or in combination with chloroquine (see, e.g., Schirmer,
H., et al., 2003, "Methylene blue as an antimalarial agent," Redox
Report, Vol. 8, pp. 272-275; Rengelshausen, J., et al., 2004,
"Pharmacokinetic interaction of chloroquine and methylene blue
combination against malaria," European Journal of Clinical
Pharmacology, Vol. 60, pp. 709-715). Malaria in humans is caused by
one of four protozoan species of the genus Plasmodium: P.
falciparum, P. vivax, P. ovale, or P. malariae. All species are
transmitted by the bite of an infected female Anopheles mosquito.
Occasionally, transmission occurs by blood transfusion, organ
transplantation, needle-sharing, or congenitally from mother to
fetus. Malaria causes 300-500 million infections worldwide and
approximately 1 million deaths annually. Drug resistance, however,
is a major concern and is greatest for P. falciparum, the species
that accounts for almost all malaria-related deaths. Drugs or drug
combinations that are currently recommended for prophylaxis of
malaria include chloroquine/proguanil hydrochloride, mefloquine,
doxycycline, and primaquine.
MTC (under the name Virostat.RTM., from Bioenvision Inc., New York)
has shown potent viricidal activity in vitro. Specifically
Virostat.RTM. is effective against viruses such as HIV and West
Nile Virus in laboratory tests. West Nile virus (WNV) is a
potentially serious illness affecting the central nervous system.
The large majority of infected people will show no visible symptoms
or mild flu-like symptoms such as fever and headache. About one in
150 will develop severe symptoms including tremors, convulsions,
muscle weakness, vision loss, numbness, paralysis, or coma.
Generally, WNV is spread by the bite of an infected mosquito, but
can also spread through blood transfusions, organ transplants,
breastfeeding or during pregnancy from mother to child.
Virostat.RTM. is also currently in clinical trials for the
treatment of chronic Hepatitis C. Hepatitis C is a viral infection
of the liver. The virus, HCV, is a major cause of acute hepatitis
and chronic liver disease, including cirrhosis and liver cancer.
HCV is spread primarily by direct contact with human blood. The
major causes of HCV infection worldwide are use of unscreened blood
transfusions, and re-use of needles and syringes that have not been
adequately sterilized. The World Health Organization has declared
hepatitis C a global health problem, with approximately 3% of the
world's population infected with HCV and it varies considerably by
region. The prevalence in the US is estimated at 1.3% or
approximately 3.5 million people. Egypt has a population of
approximately 62 million and contains the highest prevalence of
hepatitis C in the world, estimated at over 20% of the nation's
approximately 62 million people.
MTC, when combined with light, can prevent the replication of
nucleic acid (DNA or RNA). Plasma, platelets and red blood cells do
not contain nuclear DNA or RNA. When MTC is introduced into the
blood components, it crosses bacterial cell walls or viral membrane
then moves into the interior of the nucleic acid structure. When
activated with light, the compound then binds to the nucleic acid
of the viral or bacterial pathogen, preventing replication of the
DNA or RNA. Because MTC can inactivate pathogens, it has the
potential to reduce the risk of transmission of pathogens that
would remain undetected by testing.
Oral and parenteral formulations of MTC are commercially available
in the United States, usually under the name Urolene Blue.RTM..
SUMMARY OF THE INVENTION
One aspect of the invention pertains to certain compounds,
specifically, certain 3,7-diamino-10H-phenothiazine (DAPTZ)
compounds, as described herein.
Another aspect of the invention pertains to a composition
comprising a DAPTZ compound as described herein and a
pharmaceutically acceptable carrier or diluent.
Another aspect of the invention pertains to a pharmaceutical
composition comprising a DAPTZ compound as described herein and a
pharmaceutically acceptable carrier or diluent.
Another aspect of the invention pertains to a method of preparing a
pharmaceutical composition comprising admixing a DAPTZ compound as
described herein and a pharmaceutically acceptable carrier or
diluent.
Another aspect of the present invention pertains to a method of
reversing and/or inhibiting the aggregation of a protein (e.g., a
tau protein, a synuclein, etc.), for example, aggregation of a
protein associated with a neurodegenerative disease and/or clinical
dementia, comprising contacting the protein with an effective
amount of a DAPTZ compound, as described herein. Such a method may
be performed in vitro, or in vivo.
Another aspect of the present invention pertains to a method of
treatment or prophylaxis of a disease condition in a subject
comprising administering to said subject a prophylactically or
therapeutically effective amount of a DAPTZ compound, as described
herein, preferably in the form of a pharmaceutical composition.
Another aspect of the present invention pertains to a DAPTZ
compound as described herein for use in a method of treatment or
prophylaxis (e.g., of a disease condition) of the human or animal
body by therapy.
Another aspect of the present invention pertains to use of a DAPTZ
compound, as described herein, in the manufacture of a medicament
for use in the treatment or prophylaxis of a disease condition.
In one embodiment, the disease condition is a disease of protein
aggregation.
In one embodiment, the disease condition is a tauopathy, e.g., a
neurodegenerative tauopathy, e.g., Alzheimer's disease.
In one embodiment, the disease condition is skin cancer, e.g.,
melanoma.
In one embodiment, the disease condition is a viral, bacterial or
protozoal disease condition, e.g., Hepatitis C, HIV, West Nile
Virus (WNV), or malaria.
Another aspect of the present invention pertains to a method of
inactivating a pathogen in a sample (for example a blood or plasma
sample), comprising the steps of introducing a DAPTZ compound, as
described herein, into the sample, and then exposing the sample to
light.
Another aspect of the present invention pertains to a kit
comprising (a) a DAPTZ compound as described herein, preferably
provided as a pharmaceutical composition and in a suitable
container and/or with suitable packaging; and (b) instructions for
use, for example, written instructions on how to administer the
compound.
As will be appreciated by one of skill in the art, features and
preferred embodiments of one aspect of the invention will also
pertain to other aspect of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the percent reduced form (%) versus time
(minutes) for each of three compounds, B1 (MTC), B3
(N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)), and B6
(N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
iodide)), as determined using absorbance at 665 nm.
FIG. 2 is a graph of the percent reduced form (%) versus time
(minutes) for each of three compounds, B1, B3, and B6, as
determined using absorbance at 610 nm.
FIG. 3A shows the UV/visible absorption spectra for aqueous samples
of each of three compounds, B1 (open circles, maximum at 605 nm),
B3 (open squares, maximum at 660 nm), and B6 (open triangles,
maximum at 660 nm), after 20 minutes.
FIG. 3B shows the UV/visible absorption spectra for aqueous samples
of each of three compounds, B1 (open circles, maximum at 605 nm),
B3 (open squares, maximum at 605 nm), and B6 (open triangles,
maximum at 605 nm), after 3 hours.
FIG. 3C shows the UV/visible absorption spectra for aqueous samples
of each of three compounds, B1 (open circles, maximum at 605 nm),
B3 (open squares, maximum at 605 nm), and B6 (open triangles,
maximum at 605 nm), after 28 hours.
FIG. 4 shows the crystal structure of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide).
FIG. 5 shows the side-on view of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide).
FIG. 6 shows part of one helical column of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide) molecules in the crystal.
DETAILED DESCRIPTION OF THE INVENTION
Methythioninium Chloride (MTC) (also known as Methylene blue (MB);
methylthionine chloride; tetramethylthionine chloride;
3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue
9; tetramethylthionine chloride; 3,7-bis(dimethylamino)
phenazathionium chloride; Swiss blue; C.I. 52015; C.I. Solvent Blue
8; aniline violet; and Urolene Blue.RTM.) is a low molecular weight
(319.86), water soluble, tricyclic organic compound of the
following formula:
##STR00001## Methythioninium Chloride (MTC) (also known as
Methylene Blue), perhaps the most well known phenothiazine dye and
redox indicator, has also been used as an optical probe of
biophysical systems, as an intercalator in nanoporous materials, as
a redox mediator, and in photoelectrochomic imaging.
MTC, a phenothiazin-5-ium salt, may conveniently be considered to
be an "oxidized form" when considered in respect of the
corresponding 10H-phenothiazine compound,
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine, which may
conveniently be considered to be a "reduced form":
##STR00002##
The "reduced form" (the "leuko form") is known to be unstable, and
is readily and rapidly oxidized to give the corresponding
"oxidized" form.
May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp.
C1390-C1398) have shown that human erythrocytes sequentially reduce
and take up MTC; that MTC itself is not taken up by the cells; that
it is the reduced from of MTC that crosses the cell membrane; that
the rate of uptake is enzyme dependent; and that both MTC and
reduced MTC are concentrated in cells (reduced MTC re-equilibrates
once inside the cell to form MTC).
MTC and similar drugs are taken up in the gut and enter the
bloodstream. Unabsorbed drug percolates down the alimentary canal,
to the distal gut. One important undesired side-effect is the
effect of the unabsorbed drug in the distal gut, for example,
sensitisation of the distal gut and/or antimicrobial effects of the
unabsorbed drug on flora in the distal gut, both leading to
diarrhea. Therefore, it is desirable to minimize the amount of drug
that percolates to the distal gut. By increasing the drug's update
in the gut (i.e., by increasing the drug's bioavailability), dosage
may be reduced, and the undesired side-effects, such as diarrhea,
may be ameliorated.
Since it is the reduced form of MTC that is taken up by cells, it
would be desirable to administer the reduced form. This would also
reduced reliance on the rate limiting step of enzymatic
reduction.
The inventors have identified a class of compounds that may also be
considered to be in the "reduced form" when considered in respect
of MTC, and which are surprisingly and unexpectedly stable. The
compounds may therefore be described as "stabilized reduced forms,"
for example, of MTC.
These compounds are themselves active as drugs, and may also serve
as prodrugs, yielding, upon oxidation, the corresponding oxidized
compounds (e.g., MTC), which are also active as drugs.
One representative member of this class of compounds is shown
below.
##STR00003##
Another representative member of this class of compounds is shown
below.
##STR00004## The Compounds
In general, the present invention pertains certain
3,7-diamino-10H-phenothiazine compounds of the following formula
(collectively referred to herein as "diamino-phenothiazine
compounds" and/or "DAPTZ compounds"):
##STR00005## wherein: each of R.sup.1 and R.sup.9 is independently
selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl; each of R.sup.3NA and R.sup.3NB is
independently selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl,
and halogenated C.sub.1-4alkyl; each of R.sup.7NA and R.sup.7NB is
independently selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl,
and halogenated C.sub.1-4alkyl; each of HX.sup.1 and HX.sup.2 is
independently a protic acid; and pharmaceutically acceptable salts,
solvates, and hydrates thereof.
Without wishing to be bound to any particular theory, the inventors
believe that it is possible, if not likely, that the compounds
exist in the following form:
##STR00006##
Although the DAPTZ compounds are themselves salts, they may also be
provided in the form of a mixed salt (i.e., the DAPTZ in
combination with another salt). Such mixed salts are intended to be
encompassed by the term "and pharmaceutically acceptable salts
thereof". Unless otherwise specified, a reference to a particular
compound also includes salts thereof.
The DAPTZ compounds may also be provided in the form of a solvate
or hydrate. The term "solvate" is used herein in the conventional
sense to refer to a complex of solute (e.g., compound, salt of
compound) and solvent. If the solvent is water, the solvate may be
conveniently referred to as a hydrate, for example, a mono-hydrate,
a di-hydrate, a tri-hydrate, etc. Unless otherwise specified, a
reference to a particular compound also includes solvate forms
thereof.
In one embodiment, the C.sub.1-4alkyl groups are selected from:
linear C.sub.1-4alkyl groups, such as -Me, -Et, -nPr, -iPr, and
-nBu; branched C.sub.3-4alkyl groups, such as -iPr, -iBu, -sBu, and
-tBu; and cyclic C.sub.3-4alkyl groups, such as -cPr and -cBu.
In one embodiment, the C.sub.2-4alkenyl groups are selected from
linear C.sub.1-4alkenyl groups, such as --CH.dbd.CH.sub.2 (vinyl)
and --CH.sub.2--CH.dbd.CH.sub.2 (allyl).
In one embodiment, the halogenated C.sub.1-4alkyl groups are
selected from: --CF.sub.3, --CH.sub.2CF.sub.3, and
--CF.sub.2CF.sub.3.
The Groups R.sup.1 and R.sup.9
In one embodiment, each of R.sup.1 and R.sup.9 is independently
--H, -Me, -Et, or --CF.sub.3.
In one embodiment, each of R.sup.1 and R.sup.9 is independently
--H, -Me, or -Et.
In one embodiment, R.sup.1 and R.sup.9 are the same.
In one embodiment, R.sup.1 and R.sup.9 are different.
In one embodiment, each of R.sup.1 and R.sup.9 is independently
--H.
In one embodiment, each of R.sup.1 and R.sup.9 is independently
-Me.
In one embodiment, each of R.sup.1 and R.sup.9 is independently
-Et.
The Groups R.sup.3NA and R.sup.3NB
Each of R.sup.3NA and R.sup.3NB is independently selected from:
--H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
selected from: C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
-Me, -Et, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
-Me, -nPr, -nBu, --CH.sub.2--CH--CH.sub.2, or --CF.sub.3.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
-Me or -Et.
In one embodiment, R.sup.3NA and R.sup.3NB are the same.
In one embodiment, R.sup.3NA and R.sup.3NB are different.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
-Me.
In one embodiment, each of R.sup.3NA and R.sup.3NB is independently
-Et.
The Groups R.sup.7NA and R.sup.7NB
Each of R.sup.7NA and R.sup.7NB is independently selected from:
--H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
selected from: C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
-Me, -Et, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
-Me, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
-Me or -Et.
In one embodiment, R.sup.7NA and R.sup.7NB are the same.
In one embodiment, R.sup.7NA and R.sup.7NB are different.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
-Me.
In one embodiment, each of R.sup.7NA and R.sup.7NB is independently
-Et.
In one embodiment, R.sup.3NA and R.sup.3NB and R.sup.7NA and
R.sup.7NB are the same.
In one embodiment, R.sup.3NA and R.sup.3NB and R.sup.7NA and
R.sup.7NB are as defined herein, with the proviso that at least one
of R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB is other
than -Et.
Optional Provisos
In one embodiment, the compound is as defined herein, but with the
proviso that: R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB
are not each -Et.
In one embodiment, the compound is as defined herein, but with the
proviso that:
if: each of R.sup.1 and R.sup.9 is --H;
then: R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB are not
each -Et.
The Groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB)
In one embodiment:
each of R.sup.3NA and R.sup.3NB is independently C.sub.1-4alkyl,
C.sub.2-4alkenyl, or halogenated C.sub.1-4alkyl;
each of R.sup.7NA and R.sup.7NB is independently C.sub.1-4alkyl,
C.sub.2-4alkenyl, or halogenated C.sub.1-4alkyl;
optionally with the proviso that at least one of R.sup.3NA and
R.sup.3NB and R.sup.7NA and R.sup.7NB is other than -Et.
In one embodiment:
each of R.sup.3NA and R.sup.3NB is independently -Me, -Et, -nPr,
-nBu, --CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3;
each of R.sup.7NA and R.sup.7NB is independently -Me, -Et, -nPr,
-nBu, --CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3;
optionally with the proviso that at least one of R.sup.3NA and
R.sup.3NB and R.sup.7NA and R.sup.7NB is other than -Et.
In one embodiment:
each of R.sup.3NA and R.sup.3NB is independently -Me or -Et;
each of R.sup.7NA and R.sup.7NB is independently -Me or -Et;
optionally with the proviso that at least one of R.sup.3NA and
R.sup.3NB and R.sup.7NA and R.sup.7NB is other than -Et.
In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same.
In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are different.
In one embodiment, each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently selected from:
--NMe.sub.2, --NEt.sub.2, --N(nPr).sub.2, --N(Bu).sub.2, --NMeEt,
--NMe(nPr), and --N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are independently
selected from: --NMe.sub.2, --NEt.sub.2, --N(nPr).sub.2,
--N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are independently
selected from: --NMe.sub.2 and --NEt.sub.2.
In one embodiment, each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is: --NMe.sub.2.sup.+.
In one embodiment, at least one of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is other
than --NEt.sub.2.
In one embodiment, each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is other than --NEt.sub.2.
For example, in one embodiment, the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) are the
same, and are selected from: --NMe.sub.2, --N(nPr).sub.2,
--N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
The Groups HX.sup.1 and HX.sup.2
Each of HX.sup.1 and HX.sup.2 is independently a protic acid.
Examples of protic acids include, for example, inorganic acids,
such as hydrohalide acids (e.g., HCl, HBr, HI), nitric acid
(HNO.sub.3), sulphuric acid (H.sub.2SO.sub.4), and organic acids,
such as carbonic acid (H.sub.2CO.sub.3) and acetic acid
(CH.sub.3COOH).
In one embodiment, each of HX.sup.1 and HX.sup.2 is independently a
monoprotic acid.
In one embodiment, each of HX.sup.1 and HX.sup.2 is independently a
hydrohalide acid (i.e., a hydrohalic acid)
In one embodiment, each of HX.sup.1 and HX.sup.2 is independently
selected from HCl, HBr, and HI.
In one embodiment, HX.sup.1 and HX.sup.2 are the same.
In one embodiment, HX.sup.1 and HX.sup.2 are different.
In one embodiment, HX.sup.1 and HX.sup.2 are the same, and are
independently selected from HCl, HBr, and HI. In this case, the
compound (a diamino-phenothiazine compound) may conveniently be
referred to as a "diamino-phenothiazine bis(hydrogen halide)
salt".
In one embodiment, HX.sup.1 and HX.sup.2 are each HCl. In this
case, the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen chloride) salt".
In one embodiment, HX.sup.1 and HX.sup.2 are each HBr. In this
case, the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen bromide) salt".
In one embodiment, HX.sup.1 and HX.sup.2 are each HI. In this case,
the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen iodide) salt".
Some Preferred Combinations
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H, -Me, or -Et;
and
each of the groups N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H, -Me, or -Et;
and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2,
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H, -Me, or -Et;
and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2; and
each of HX.sup.1 and HX.sup.2 is independently selected from HCl,
HBr, and HI.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H, -Me, or -Et;
and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
each of HX.sup.1 and HX.sup.2 is independently selected from HCl,
HBr, and HI.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2; and
each of HX.sup.1 and HX.sup.2 is independently selected from HCl,
HBr, and HI.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
each of HX.sup.1 and HX.sup.2 is independently selected from HCl,
HBr, and HI.
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
each of HX.sup.1 and HX.sup.2 is HCl.
##STR00007##
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
each of HX.sup.1 and HX.sup.2 is HBr.
##STR00008##
In one embodiment:
each of R.sup.1 and R.sup.9 is independently --H; and
each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
each of HX.sup.1 and HX.sup.2 is HI.
##STR00009## Isotopic Variation
In one embodiment, one or more of the carbon atoms of the compound
is .sup.11C, .sup.13C, or .sup.14C.
In one embodiment, one or more of the carbon atoms of the compound
is .sup.11C.
In one embodiment, one or more of the carbon atoms of the compound
is .sup.13C.
In one embodiment, one or more of the carbon atoms of the compound
is .sup.14C.
In one embodiment, one or more of the nitrogen atoms of the
compound is .sup.15N.
In one embodiment, one or more or all of the carbon atoms of one or
more or all of the groups R.sup.3NA, R.sup.3NB, R.sup.7NA,
R.sup.7NB, R.sup.1, R.sup.9, and R.sup.10 is .sup.11C. (Or
.sup.13C.) (Or .sup.14C.)
In one embodiment, one or more or all of the carbon atoms of one or
more or all of the groups R.sup.3NA, R.sup.3NB, R.sup.7NA, and
R.sup.7NB is .sup.11C. (Or .sup.13C.) (Or .sup.14C.)
In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are:
--N(.sup.11CH.sub.3).sub.2. (Or --N(.sup.13CH.sub.3).sub.2.) (Or
--N(.sup.14CH.sub.3).sub.2.)
Compatible Combinations
All compatible combinations of the embodiments described above are
explicitly disclosed herein as if each combination was specifically
and individually recited.
Some Preferred Embodiments
In one embodiment, the compound is selected from the following
compounds, and pharmaceutically acceptable salts, solvates, and
hydrates thereof.
##STR00010## ##STR00011##
In one embodiment, the compound is selected from the following
compounds, and pharmaceutically acceptable salts, solvates, and
hydrates thereof.
##STR00012## ##STR00013##
In one embodiment, the compound is selected from the following
compounds, and pharmaceutically acceptable salts, solvates, and
hydrates thereof.
##STR00014## ##STR00015## Purity
The DAPTZ compounds of the present invention may conveniently be
described as being in a "stabilized reduced form". The compounds
oxidize (e.g., autoxidize) to give the corresponding oxidized
forms. Thus, it is likely, if not inevitable, that compositions
comprising the DAPTZ compounds of the present invention will
contain, as an impurity, as least some of the corresponding
oxidized compound.
Thus, another aspect of the present invention pertains to DAPTZ
compounds, as described herein, in substantially purified form
and/or in a form substantially free from contaminants (e.g., the
corresponding oxidized compound, other contaminants).
In one embodiment, the substantially purified form is at least 50%
by weight pure, e.g., at least 60% by weight pure, e.g., at least
70% by weight pure, e.g., at least 80% by weight pure, e.g., at
least 90% by weight pure, e.g., at least 95% by weight pure, e.g.,
at least 97% by weight pure, e.g., at least 98% by weight pure,
e.g., at least 99% by weight pure.
In one embodiment, the contaminants represent no more than 50% by
weight, e.g., no more than 40% by weight, e.g., no more than 30% by
weight, e.g., no more than 20% by weight, e.g., no more than 10% by
weight, e.g., no more than 5% by weight, e.g., no more than 3% by
weight, e.g., no more than 2% by weight, e.g., no more than 1% by
weight.
Product-by-Process
In one embodiment, the DAPTZ compound is one which is obtained by,
or is obtainable by, a method as described herein.
Chemical Synthesis
Methods for the chemical synthesis of DAPTZ compounds of the
present invention are described herein. These and/or other well
known methods may be modified and/or adapted in known ways in order
to facilitate the synthesis of additional DAPTZ compounds within
the scope of the present invention.
For example, a suitable phenothiazine may be converted to the
corresponding 3,7-dinitro-phenothiazine, for example, using sodium
nitrite with acetic acid and chloroform. The ring amino group may
then be protected, for example, as the acetate, for example, using
acetic anhydride and pyridine. The nitro groups may then be reduced
to amino groups, for example, using tin (II) chloride with ethanol.
The amino groups may then be substituted, for example,
disubstituted, for example, methyl disubstituted, for example,
using methyl iodide, sodium hydroxide, DMSO, and tetra-n-butyl
ammonium bromide. The amino group may then be deprotected, for
example, the N-acetyl group may be removed, for example, using
concentrated aqueous hydrochloride acid. The corresponding salt is
then prepared, for example, using concentrated aqueous hydrochloric
acid, for example, at the same time as deprotection. An example of
such a method is illustrated in the following scheme.
##STR00016##
Thus, another aspect of the invention pertains to a method of
preparing a 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the
following formula:
##STR00017## wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB,
R.sup.7NA, R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein
(for example, where HX.sup.1 and HX.sup.2 are each HCl), comprising
the step of: (vi) salt formation (SF).
In one embodiment, the method comprises the steps of: (v) ring
amino deprotection (DP); and (vi) salt formation (SF).
In one embodiment, the method comprises the steps of: (iv) amine
substitution (AS), optional (v) ring amino deprotection (DP), and
(vi) salt formation (SF).
In one embodiment, the method comprises the steps of (iii) nitro
reduction (NR), (iv) amine substitution (AS), (v) ring amino
deprotection (DP), and (vi) salt formation (SF).
In one embodiment, the method comprises the steps of optional (ii)
ring amino protection (AP), (iii) nitro reduction (NR), (iv) amine
substitution (AS), (v) ring amino deprotection (DP), and (vi) salt
formation (SF).
In one embodiment, the method comprises the steps of (i) nitration
(NO), (ii) ring amino protection (AP), (iii) nitro reduction (NR),
(iv) amine substitution (AS), (v) ring amino deprotection (DP), and
(vi) salt formation (SF).
In one embodiment, the steps are performed in the order listed
(i.e., any step in the list is performed at the same time as, or
subsequent to, the preceding step in the list).
In one embodiment, the step of (v) ring amino deprotection (DP) and
the step of (vi) salt formation (SF) are performed simultaneously
(i.e., as one step).
In one embodiment, the nitration (NO) step is: (i) nitration (NO),
wherein a 10H-phenothiazine is converted to a
3,7-dinitro-10H-phenothiazine, for example:
##STR00018##
In one embodiment, nitration is performed using a nitrite, for
example, sodium nitrite, for example, sodium nitrite with acetic
acid and chloroform. In one embodiment, R.sup.10 is --H.
In one embodiment, the ring amino protection (AP) step is: (ii)
ring amino protection (AP), wherein the ring amino group (--NH--)
of a 3,7-dinitro-10H-phenothiazine is converted to a protected ring
amino group (--NR.sup.prot), for example:
##STR00019##
In one embodiment, ring amino protection is achieved as an acetate,
for example, using acetic anhydride, for example, using acetic
anhydride and pyridine.
In one embodiment, the nitro reduction (NR) step is: (iii) nitro
reduction (NR), wherein each of the nitro (--NO.sub.2) groups of a
protected 3,7-dinitro-10H-phenothiazine is converted to an amino
(--NH.sub.2) group, for example:
##STR00020##
In one embodiment, nitro reduction may be performed using, for
example, tin (II) chloride, for example, tin (II) chloride with
ethanol.
In one embodiment, the amine substitution (AS) step is: (iv) amine
substitution (AS), wherein each of the amino (--NH.sub.2) groups of
a protected 3,7-diamino-10H-phenothiazine is converted to
disubstituted amino group, for example:
##STR00021##
In one embodiment, amine substitution is performed using an alkyl
halide, for example, an alkyl iodide, for example, methyl iodide,
for example, methyl iodide with sodium hydroxide, DMSO, and
tetra-n-butyl ammonium bromide.
In one embodiment, the ring amino deprotection (DP) step is: (v)
ring amino deprotection (DP), wherein the protecting group,
R.sup.Prot, is removed, for example:
##STR00022##
In one embodiment, ring amino deprotection may be performed using
acid, for example, hydrochloric acid, for example, concentrated
aqueous hydrochloric acid.
In one embodiment, the step is: (vi) salt formation (SF), wherein
the corresponding salt is formed, for example:
##STR00023##
In one embodiment, salt formation may be performed using acid, for
example, hydrochloric acid, for example, concentrated aqueous
hydrochloric acid.
In one embodiment, the steps of ring amine deprotection and salt
formation are performed simultaneously (i.e., as one step), for
example, compound (1) is formed from compound (2) in one step.
In another approach, a suitable thioninium chloride (for example,
methylthioninium chloride, MTC, also known as Methylene Blue) is
converted to the corresponding halide, for example, by reaction
with potassium iodide, for example, aqueous potassium iodide. The
resulting thioninium iodide is then reduced, for example, with
ethyl iodide and ethanol, and the corresponding salt formed. A
similar method is described in Drew, H. D. K, and Head, F. S. H.,
"Derivatives of Methylene-blue," Journal of the Chemical Society,
1933, pp. 248-253. An example of such a method is illustrated in
the following scheme.
##STR00024##
Thus, another aspect of the invention pertains to a method of
preparing a 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the
following formula:
##STR00025## wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB,
R.sup.7NA, R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein
(for example, where HX.sup.1 and HX.sup.2 are each HI), comprising
the step of: (ii) reduction and iodide salt formation (RISF).
In one embodiment, the method comprises the steps of: (i) iodide
exchange (IE); and (ii) reduction and iodide salt formation
(RISF).
In one embodiment, the steps are performed in the order listed
(i.e., any step in the list is performed at the same time as, or
subsequent to, the preceding step in the list).
In one embodiment, the iodide exchange (IE) step is: (i) iodide
exchange (IE), wherein a 3,7-di(disubstituted amino)-thioninium
salt is converted to the corresponding 3,7-di(disubstituted
amino)-thioninium iodide, for example (where Y.sup.- is an anionic
counter ion, for example, halide, for example, chloride or
bromide):
##STR00026##
In one embodiment, iodide exchange (IE) is achieved by reaction
with potassium iodide, for example, aqueous potassium iodide.
In one embodiment, the reduction and iodide salt formation (RISF)
step is: (ii) reduction and iodide salt formation (RISF), wherein a
3,7-di(disubstituted amino)-thioninium iodide is reduced and
converted to the corresponding 3,7-diamino-10H-phenothiazine iodide
compound, for example:
##STR00027##
In one embodiment, reduction and iodide salt formation (RISF) is
achieved by reaction with ethyl iodide, for example, ethyl iodide
and ethanol.
In another approach, an appropriate thioninium salt, for example,
ethyl thioninium semi zinc chloride, is simultaneously reduced and
the ring amino group protected, for example, by reaction with
phenylhydrazine, ethanol, acetic anhydride, and pyridine. The
corresponding salt may then be prepared, for example, using
concentrated aqueous hydrochloric acid, for example, at the same
time as deprotection. An example of such a method is illustrated in
the following scheme.
##STR00028##
Thus, another aspect of the invention pertains to a method of
preparing a 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the
following formula:
##STR00029## wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB,
R.sup.7NA, R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein
(for example, where HX.sup.1 and HX.sup.2 are each HI), comprising
the step of: comprising the step of: (iv) salt formation (SF).
In one embodiment, the method comprises the steps of (iii) ring
amino deprotection (DP), and (iv) salt formation (SF).
In one embodiment, the method comprises the steps of (ii) ring
amino protection (AP), (iii) ring amino deprotection (DP), and (iv)
salt formation (SF).
In one embodiment, the method comprises the steps of (i) reduction
(RED) (ii) ring amino protection (AP), (iii) ring amino
deprotection (DP), and (iv) salt formation (SF).
In one embodiment, the steps are performed in the order listed
(i.e., any step in the list is performed at the same time as, or
subsequent to, the preceding step in the list).
In one embodiment, the step of (i) reduction (RED) and the step of
(ii) ring amino protection (AP) are performed simultaneously (i.e.,
as one step).
For example, in one embodiment, the combined reduction (RED) step
and ring amino protection (AP) step is: (i) reduction (RED) and
ring amino protection (AP), wherein a 3,7-di(disubstituted
amino)-thioninium salt is reduced to give the corresponding
3,7-di(disubstituted amino)-10H-phenothiazine, and the ring amino
group (--NH--) of the 3,7-di(disubstituted amino)-10H-phenothiazine
is converted to a protected ring amino group (--R.sup.prot) to give
the corresponding protected 3,7-di(disubstituted
amino)-10H-phenothiazine, for example:
##STR00030##
In one embodiment, Y represents Cl.sup.-.
In one embodiment, the combined reduction (RED) step and ring amino
protection (AP) step is achieved using phenylhydrazine and acetic
anhydride, for example, phenylhydrazine, ethanol, acetic anhydride,
and pyridine.
In one embodiment, the step of (iii) ring amino deprotection (DP)
and the step of (iv) salt formation (SF) are performed
simultaneously (i.e., as one step).
For example, in one embodiment, the combined ring amino
deprotection (DP) step and salt formation (SF) step is: (ii) ring
amino deprotection (DP) and salt formation (SF), wherein the
protecting group of a protected 3,7-di(disubstituted
amino)-10H-phenothiazine is removed to give a 3,7-di(disubstituted
amino)-10H-phenothiazine, and the corresponding salt is formed, for
example:
##STR00031##
In one embodiment, the combined ring amino deprotection (DP) step
and salt formation (SF) step may be performed using acid, for
example, hydrochloric acid, for example, concentrated aqueous
hydrochloric acid.
In a similar approach, an appropriate thioninium chloride (e.g.,
methyl thioninium chloride, ethyl thioninium chloride) is first
reduced and acetylated to give the corresponding
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone, for example,
by reaction with hydrazine (NH.sub.2NH.sub.2), methyl hydrazine
(MeNHNH.sub.2), or sodium borohydride (NaBH.sub.4); and acetic
anhydride ((H.sub.3CCO).sub.2O); for example, in the presence of a
suitable base, for example, pyridine (C5H5N) or Hunig's base
(diisopropylethylamine, C.sub.8H.sub.19N), for example, in a
suitable solvent, for example, ethanol or acetonitrile. The reduced
and acetylated compound is then deprotected (by removing the acetyl
group), for example, by reaction with a suitable halic acid, for
example, hydrochloric acid or hydrobromic acid, in a suitable
solvent, for example, ethanol, and optionally with the addition of
a suitable ether, for example, diethyl ether.
Compositions
Another aspect of the invention pertains to a composition
comprising a DAPTZ compound, as described herein, and a
pharmaceutically acceptable carrier or diluent.
Uses
Reversing and/or Inhibiting the Aggregation of a Protein
One aspect of the invention is the use of a DAPTZ compound, as
described herein, to regulate (e.g., to reverse and/or inhibit) the
aggregation of a protein, for example, aggregation of a protein
associated with a neurodegenerative disease and/or clinical
dementia. The aggregation may be in vitro, or in vivo, and may be
associated with a disease state as discussed below.
Thus, one aspect of the invention pertains to a method of
regulating (e.g., reversing and/or inhibiting) the aggregation of a
protein, for example, aggregation of a protein associated with a
neurodegenerative disease and/or clinical dementia, comprising
contacting the protein with an effective amount of a DAPTZ
compound, as described herein. The method may be performed in
vitro, or in vivo.
Similarly, one aspect of the invention pertains to a method of
regulating (e.g., reversing and/or inhibiting) the aggregation of a
protein in the brain of a mammal, which aggregation is associated
with a disease state as described herein, the treatment comprising
the step of administering to said mammal in need of said treatment,
a prophylactically or therapeutically effective amount of a DAPTZ
compound, as described herein, that is an inhibitor of said
aggregation.
Methods of Treatment
Another aspect of the present invention pertains to a method of
treatment comprising administering to a patient in need of
treatment a prophylactically or therapeutically effective amount of
a DAPTZ compound, as described herein, preferably in the form of a
pharmaceutical composition.
Use in Methods of Therapy
Another aspect of the present invention pertains to a DAPTZ
compound, as described herein, for use in a method of treatment
(e.g., of a disease condition) of the human or animal body by
therapy.
Use in the Manufacture of Medicaments
Another aspect of the present invention pertains to use of a DAPTZ
compound, as described herein, in the manufacture of a medicament
for use in treatment (e.g., of a disease condition).
In one embodiment, the medicament comprises the DAPTZ compound.
Disease Conditions Treated--Diseases of Protein Aggregation
The DAPTZ compounds of the present invention are useful in the
treatment or prophylaxis of diseases of protein aggregation.
Thus, in one embodiment, the disease condition is a disease of
protein aggregation, and, for example, the treatment is with an
amount of a DAPTZ compound, as described herein, sufficient to
inhibit the aggregation of the protein associated with said disease
condition.
In general, the protein aggregation is that which arises from an
induced conformational polymerisation interaction, i.e., one in
which a conformational change of the protein, or in a fragment
thereof, gives rise to templated binding and aggregation of further
(precursor) protein molecules in a self-propagating manner. Once
nucleation is initiated, an aggregation cascade may ensue which
involves the induced conformational polymerisation of further
protein molecules, leading to the formation of toxic product
fragments in aggregates which are substantially resistant to
further proteolysis. The protein aggregates thus formed are thought
to be a proximal cause of disease states manifested as
neurodegeneration, clinical dementia, and other pathological
symptoms.
The following Table provides a listing of various
disease-associated aggregating proteins and the corresponding
diseases of protein aggregation.
TABLE-US-00001 Diseases of protein aggregation Aggregating Fibril
domain and/or subunit Protein Disease mutations size (kDa)
Reference Neuro-degenerative disorders Prion protein Prion diseases
Inherited and 27 Prusiner (1998) sporadic forms (CJD, nvCJD, Fatal
PrP-27-30; many familial insomnia, mutations. Gerstmann-Straussler-
Scheinker syndrome, Kuru) Fibrillogenic Gasset et al. domains: 113-
(1992) 120, 178-191, 202-218. Tau protein Alzheimer's disease,
Inherited and 10-12 Wischik et al. Down's syndrome, sporadic forms
(1988) FTDP-17, CBD, post- encephalitic parkinsonism, Pick's
disease, parkinsonism with dementia complex of Guam Truncated tau
(tubulin-binding domain) 297-391. Mutations in tau Hutton et al. in
FTDP-17. (1998) Many mutations Czech et al. in presenilin (2000)
proteins. Amyloid Alzheimer's disease, Inherited and 4 Glenner
& .beta.-protein Down's syndrome sporadic forms Wong, (1984)
Amyloid .beta.- protein; 1-42(3). 11 mutations in Goate et al. APP
in rare (1991) families. Huntingtin Huntington's disease N-termini
of 40 DiFiglia et al. protein with (1997) expanded glutamine
repeats. Ataxins Spinocerebellar ataxias Proteins with Paulson et
al. (1, 2, 3, 7) (SCA1, 2, 3, 7) expanded (1999) glutamine repeats.
Atrophin Dentarubropallidoluysian Proteins with Paulson et al.
atrophy (DRPLA) expanded (1999) glutamine repeats. Androgen Spinal
and bulbar Proteins with Paulson et al. receptor muscular atrophy
expanded (1999) glutamine repeats. Neuroserpin Familial
encephalopathy Neuroserpin; 57 Davis et al. with neuronal inclusion
S49P, S52R. (1999) bodies (FENIB) .alpha.-Synuclein Parkinson's
disease, Inherited and 19 Spillantini et al. dementia with Lewy
bodies, sporadic forms (1998) multiple system atrophy A53T, A30P in
Polymeropoulos rare autosomal- et al. (1997) dominant PD families.
Cystatin C Hereditary cerebral Cystatin C less 12-13 Abrahamson et
angiopathy (Icelandic) 10 residues; al. (1992) L68Q. Superoxide
Amyotrophic lateral SOD1 mutations. Shibata et al. dismutase 1
sclerosis (1996) Non-neuro-degenerative disorders Haemoglobin
Sickle cell anaemia Haemoglobin Carrell & beta chain (S).
Gooptu (1998) Inclusion body haemolysis Many mutations. Serpins
.alpha.1-Antitrypsin deficiency Mutations Lomas et al. (emphysema,
cirrhosis) (1992) Antithrombin deficiency Mutatons Carrell &
(thromboembolic disease) Gooptu (1998) C1-inhibitor deficiency
Mutations Carrell & (angioedema) Gooptu (1998) Immunoglobulin
Plasma cell dyscrasias light chain or 0.5-25 Westermark et light
chain (primary systemic AL fragments. al. (1985) amyloidosis) Serum
amyloid Reactive, secondary 76-residue 4.5-7.5 Westermark et A
systemic AA amyloidosis fragment (critical al. (1985) residues
2-12). Chronic inflammatory disease Transthyretin Familial amyloid
Tetramer 10-14 Gustavsson et polyneuropathy dissociated to al.
(1991) (systemic; FAP I) conformational monomer variant. Many
mutations (some not associated with amyloid; several different
types of disease). Senile cardiac Normal 10-14 Gustavsson et
amyloidosis transthyretin al. (1991) Gelsolin Familial amyloidosis
- D187Q leads to 9.5 Maury & Finnish type (FAP IV) truncated
173- Baumann 225/243 (critical (1990) residues 182- 192). .beta.2-
Haemodialysis .beta.2-Microglobulin 12-25 Gorevic et al.
Microglobulin amyloidosis (1985) Prostatic amyloid Apolipoprotein
Familial amyloid N-terminal 83-93 9 Booth et al. Al polyneuropathy
residues; G26R, (1997) (systemic; FAP III) W50R, L60R Lysozyme
Familial visceral Lysozyme or 14 Pepys et al. amyloidosis fragments
(with (1993) or without I56T, D67H) Amylin (Islet Type II diabetes
Fragments 3.9 Westermark amyloid (NIDDM) (critical core of (1990)
polypeptide) 20-29); no mutations Fibrinogen Hereditary renal
Fibrinogen 7-10 Uemichi et al. .alpha.-chain amyloidosis fragments
(1992) Procalcitonin Medullary carcinoma of Calcitonin 3.4 Sletten
et al. thyroid fragments (1976) Atrial natriuretic Cardiac
amyloidosis ANF, no mutants 3.5 Johansson et factor al. (1987)
Insulin Injection localised Insulin Dische et al. amyloidosis
(1988) Other proteins (in vitro) Other proteins Chiti et al.
forming amyloid (1999)
REFERENCES FOR THE ABOVE TABLE
Abrahamson, M., Jonsdottir, S., Olafsson, I. & Grubb, A. (1992)
Hereditary cystatin C amyloid angiopathy identification of the
disease-causing mutation and specific diagnosis by polymerase chain
reaction based analysis. Human Genetics 89, 377-380. Booth, D. R.,
Sunde M., Bellotti, V., Robinson, C. V., Hutchinson, W. L., Fraser,
P. E., Hawkins, P. N., Dobson, G. M., Radford, S. E., Blake, C. C.
F. & Pepys, M. B. (1997) Instability, unfolding and aggregation
of human lysozyme variants underlying amyboid fibrilbogenesis.
Nature 385, 787-793. Carrell, R. W. & Gooptu, B. (1998)
Conformational changes and disease--serpins, prions and Alzheimers.
Current Opinion in Structural Biology 8, 799-809. Chiti, F.,
Webster, P., Taddei, N., Clark, A., Stafani, M., Ramponi, C. &
Dobson, C. (1999) Designing conditions for in vitro formation of
amyloid protofilaments and fibrils. Proceedings of the National
Academy of Sciences, USA 96, 3590-3594. Czech, C., Tremp, G. &
Pradier, L. (2000) Presenilins and Alzheimer's disease: biological
functions and pathogenic mechanisms. Progress in Neurobiology 60,
363-384. Davis, R. L., Shrimpton, A. E., Holohan, P. D., Bradshaw,
C., Feiglin, D., Collins, G. H., Sonderegger, P., Kinter, J.,
Becker, L. M., Lacbawan, F., Krasnewich, D., Muenke, M., Lawrence,
D. A., Yerby, M. S., Shaw, C.-M., Gooptu, B., Elliott, P. R.,
Finch, J. T., Carrell, R. W. & Lomas, D. A. (1999) Familial
dementia caused by polymerization of mutant neuroserpin. Nature
401, 376-379. DiFiglia M., Sapp, E., Chase, K. O., Davies, S. W.,
Bates, G. P., Vonsattel, J. P. & Aronin, N. (1997) Aggregation
of huntingtin in neuronal intranuclear inclusions and dystrophic
neurites in brain. Science 277, 1990-1993. Dische, F. E.,
Wernstedt, C., Westermark, G. T., Westermark, P., Pepys, M. B.,
Rennie, J. A., Gibbey, S. G. & Watkins, P. J. (1988) Insulin as
an amyloid-fibril protein at sites of repeated insulin injections
in a diabetic patient. Diabetologia 31, 158-161. Gasset, M.,
Bladwin, M. A., Lloyd, D. H., abriel, J.-M., Holtzman, D. M.,
Cohen, F. E., Fletterick, R. & Prusiner, S. B. (1992) Predicted
a-helical region of the prion protein when synthesized as peptides
form amyboid. Proceedings of the National Academy of Sciences, USA
89, 10940-10944. Glenner, G. G. & Wong, C. W. (1984)
Alzheimer's disease: initial report of the purification and
characterisation of a novel cerebrovascular amyloid protein.
Biochemical and Biophysical Research Communications 120, 885-890.
Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J., Crawford,
F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L.,
Mant, R., Newton, P., Rooke, K., Roques, P., Talbot, C.,
Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M.
& Hardy, J. (1991) Segregation of a missense mutation in the
amyboid precursor protein gene with familial Aizheimer's disease.
Nature 349, 704-706. Gorevic, P. D., Casey, T. T., Stone, W. J.,
DiRaimondo, C. R., Prelli, F. C. & Frangione, B. (1985) b-2
Microgiobulin is an amyboidogenic protein in man. Journal of
Clinical Investigation 76, 2425-2429. Gustavsson, A., EngstrOm, U.
& Westermark, P. (1991) Normal transthyretin and synthetic
transthyretin fragments form amyloid-like fibrils in vitro.
Biochemical and Biophysical Research Communications 175, 1159-1164.
Hutton, M., Lendon, C., Rizzu, P., Baker, M., Froelich, S.,
Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A.,
Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D.,
Davies P., Petersen, R. C., Stevens, M., de Graaf, E., Wauters, E.,
van Baren, J., Hillebrand, M., Joosse, M., Kwon, J. M., Nowotny,
P., Che, L. K., Norton, J., Morris, J. C., Reed, L. A.,
Trojanowski, J. Q., Basun, H., Lannfelt, L., Neystat, M., Fahn, S.,
Dark, F., Tannenberg, T., Dodd, P. R., Hayward, N., Kwok, J. B. J.,
Schofield, P. R., Andreadis, A., Snowden, J., Craufurd, D., Neary,
D., Owen, F., Oostra, B A., Hardy, J., Goate, A., van Swieten, J.,
Mann, D., Lynch, T. & Heutink, P. (1998) Association of
missense and 5'-splice-site mutations in tau with the inherited
dementia FTDP-17. Nature 393, 702-705. Johansson, B., Wernstedt, C.
& Westermark, P. (1987) Atrial natriuretic peptide deposited as
atrial amyloid fibrils. Biochemical and Biophysical Research
Communications 148, 1087-1092. Lomas, D. A., Evans, D. L., Finch,
J. T. & Carrell, R. W. (1992) The mechanism of Z a1-antitrypsin
accumulation in the liver. Nature 357, 605-607. Maury, C. P. &
Baumann, M. (1990) Isolation and characterization of cardiac
amyloid in familial amyloid polyneuropathy type IV (Finnish):
relation of the amyloid protein to variant gelsolin. Biochimica et
Biophysica Acta 1096, 84-86. Paulson, H. L. (1999) Human genetics
'99: trinucleotide repeats. American Journal of Human Genetics 64,
339-345. Pepys, M. B., Hawkins, P. N., Booth, D. R., Vigushin, D.
M., Tennent, G. A., Soutar, A. K., Totty, N., Nguyen, D., Blake, C.
C. F., Terry, C. J., Feast, T. G., Zalin, A. M. & Hsuan, J. J.
(1993) Human lysozyme gene mutations cause hereditary systemic
amyloidosis. Nature 362, 553-557. Polymeropoulos, M. H., Lavedan,
C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root,
H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa,
S., Athanassiadou, A., Papaetropoulos, T., Johnson, W. G.,
Lazzarini, A. M., Duvoisin, R. C., Di lorio, G., Golbe, L. I. &
Nussbaum, R. L. (1997) Mutation in the a-synuclein gene identified
in families with Parkinson's disease. Science 276, 2045-2047.
Prusiner, S. B., Scott, M. R., DeArmorid, S. J. & Cohen, F. E.
(1998) Prion protein biology. Cell 93, 337-348. Shibata, N.,
Hirano, A., Kobayashi, M., Siddique, T., Deng, H. X., Hung, W. Y.,
Kato, T. & Asayama, K. (1996) Intense superoxide dismutase-1
immunoreactivity in intracytoplasmic hyaline inclusions of familial
amyotrophic lateral sclerosis with posterior column involvement.
Journal of Neuropathology and Experimental Neurology 55, 481-490.
Sletten, K., Westermark, P. & Natvig, J. B. (1976)
Characterization of amyloid fibril proteins from medullary
carcinoma of the thyroid. Journal of Experimental Medicine 143,
993-998. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa,
M. & Goedert, M. (1998) a-Synuclein in filamentous inclusions
of Lewy bodies from Parkinson's disease and dementia with Lewy
bodies. Proceedings of the National Academy of Sciences, USA 95,
6469-6473. Uemichi, I., Liuepnicks, J. j. & Benson, M. D.
(1994) Hereditary renal amyloidosis with a novel variant
fibrinogen. Journal of Clinical Investigation 93, 731-736.
Westermark, P., Engstrom, U., Johnson, K. H., Westermark, G. T.
& Betsholtz, C. (1990) Islet amyloid polypeptide: pinpointing
amino acid residues linked to amyloid fibril formation. Proceedings
of the National Academy of Sciences, USA 87, 5036-5040. Westermark,
P., Johnson, K. H., O'Brien, T O. & Betsholtz, C. (1992) Islet
amyloid polypeptide--a novel controversy in diabetes research.
Diabetologia 35, 297-303. Westerrnark, P., Johnson, K. H. &
Pitkanen, P. (1985) Systemic amyloidosis: A review with emphasis on
pathogenesis. Applied Physiology 3, 55-68. Wischik, C. M., Novak,
M., Thogersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R.,
Walker, J. E., Milstein, C., M., R. & Klug, A. (1988) Isolation
of a fragment of tau derived from the core of the paired helical
filament of Alzheimer's disease. Proceedings of the National
Academy of Sciences, USA 85, 4506-4510.
As described in WO 02/055720 and U.S. patent application No.
60/786,700 filed on 29 Mar. 2006 (title: Inhibitors of Protein
Aggregation), diaminophenothiazines have utility in the inhibition
of such protein aggregating diseases.
Thus it will be appreciated that, except where context requires
otherwise, description of embodiments with respect to tau protein
or tau-like proteins (e.g., MAP2), should be taken as applying
equally to the other proteins discussed herein (e.g.,
.beta.-amyloid, synuclein, prion, etc.) or other proteins which may
initiate or undergo a similar pathological aggregation by virtue of
conformational change in a domain critical for propagation of the
aggregation, or which imparts proteolytic stability to the
aggregate this formed (see, e.g., the article by Wischik et al. in
"Neurobiology of Alzheimer's Disease", 2nd Edition, 2000, Eds.
Dawbarn, D. and Allen, S. J., The Molecular and Cellular
Neurobiology Series, Bios Scientific Publishers, Oxford). All such
proteins may be referred to herein as "aggregating disease
proteins."
Likewise, where mention is made herein of "tau-tau aggregation", or
the like, this may also be taken to be applicable to other
"aggregating-protein aggregation", such as .beta.-amyloid
aggregation, prion aggregation, synuclein aggregation, etc. The
same applies for "tau proteolytic degradation" etc.
Preferred Aggregating Disease Proteins
Preferred embodiments of the invention are based on tau protein.
The term "tau protein," as used herein, refers generally to any
protein of the tau protein family. Tau proteins are characterised
as being one among a larger number of protein families which
co-purify with microtubules during repeated cycles of assembly and
disassembly (see, e.g., Shelanski et al., 1973, Proc. Natl. Acad.
Sci. USA, Vol. 70, pp. 765-768), and are known as
microtubule-associated-proteins (MAPs). Members of the tau family
share the common features of having a characteristic N-terminal
segment, sequences of approximately 50 amino acids inserted in the
N-terminal segment, which are developmentally regulated in the
brain, a characteristic tandem repeat region consisting of 3 or 4
tandem repeats of 31-32 amino acids, and a C-terminal tail.
MAP2 is the predominant microtubule-associated protein in the
somatodendritic compartment (see, e.g., Matus, A., in
"Microtubules" [Hyams and Lloyd, Eds.] pp. 155-166, John Wiley and
Sons, New York, USA). MAP2 isoforms are almost identical to tau
protein in the tandem repeat region, but differ substantially both
in the sequence and extent of the N-terminal domain (see, e.g.,
Kindler and Garner, 1994, Mol. Brain Res., Vol. 26, pp. 218-224).
Nevertheless, aggregation in the tandem-repeat region is not
selective for the tau repeat domain. Thus it will be appreciated
that any discussion herein in relation to tau protein or tau-tau
aggregation should be taken as relating also to tau-MAP2
aggregation, MAP2-MAP2 aggregation, and so on.
In one embodiment, the protein is tau protein.
In one embodiment, the protein is a synuclein, e.g., .alpha.- or
.beta.-synuclein.
Where the protein is tau protein, in one embodiment of the present
invention, there is provided a method of inhibiting production of
protein aggregates (e.g. in the form of paired helical filaments
(PHFs), optionally in neurofibrillary tangles (NFTs) in the brain
of a mammal, the treatment being as described above.
Preferred Diseases of Protein Aggregation
Notably it is not only Alzheimer's disease (AD) in which tau
protein (and aberrant function or processing thereof) may play a
role. The pathogenesis of neurodegenerative disorders such as
Pick's disease and Progressive Supranuclear Palsy (PSP) appears to
correlate with an accumulation of pathological truncated tau
aggregates in the dentate gyrus and stellate pyramidal cells of the
neocortex, respectively. Other dementias include fronto-temporal
dementia (FTD); parkinsonism linked to chromosome 17 (FTDP-17);
disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);
pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;
pallido-nigro-luysian degeneration (PNLD); cortico-basal
degeneration (CBD) and others (see, e.g., the article by Wischik et
al. in "Neurobiology of Alzheimer's Disease", 2nd Edition, 2000,
Eds. Dawbarn, D. and Allen, S. J., The Molecular and Cellular
Neurobiology Series, Bios Scientific Publishers, Oxford; especially
Table 5.1). All of these diseases, which are characterized
primarily or partially by abnormal tau aggregation, are referred to
herein as "tauopathies".
Thus, in one embodiment, the disease condition is a tauopathy.
In one embodiment, the disease condition is a neurodegenerative
tauopathy.
In one embodiment, the disease condition is Alzheimer's
disease.
In one embodiment, treatment (e.g., treatment of a
neurodegenerative tauopathy, e.g., Alzheimer's disease) may
optionally be in combination with one or more other agents, for
example, one or more cholinesterase inhibitors (such as Donepezil
(also known as Aricept.TM.), Rivastigmine (also known as
Exelon.TM.), Galantamine (also known as Reminyl.TM.), NMDA receptor
antagonists (such as Memantine (also known as Ebixa.TM.,
Namenda.TM.), muscarinic receptor agonists, and/or inhibitors of
amyloid precursor protein processing that leads to enhanced
generation of beta-amyloid.
Disease Conditions Treated--Other Disease Conditions
In one embodiment, the disease condition is skin cancer.
In one embodiment, the disease condition is melanoma.
In one embodiment, the disease condition is a viral, bacterial or
protozoal disease condition.
In one embodiment, the (protozoal) disease condition is
malaria.
In this embodiment, treatment may be in combination with one or
more antimicrobial agents, for example, chloroquine and/or
atovaquone.
In one embodiment, the (viral) disease condition is caused by
Hepatitis C, HIV, or West Nile Virus (WNV).
Other Uses
Another aspect of the present invention pertains to use of a DAPTZ
compound, as described herein, in a method of inactivating a
pathogen in a sample (for example a blood or plasma sample),
comprising the steps of introducing the DAPTZ compound into the
sample, and exposing the sample to light.
For example, in one embodiment, the method comprises the steps of
introducing the DAPTZ compound into the sample, and then exposing
the sample to light.
Use as Ligands
The DAPTZ compounds that are capable of inhibiting the aggregation
of tau protein will also be capable of acting as ligands or labels
of tau protein (or aggregated tau protein). Thus, in one
embodiment, the DAPTZ compound is a ligand of tau protein (or
aggregated tau protein).
Such DAPTZ compounds (ligands) may incorporate, be conjugated to,
be chelated with, or otherwise be associated with, other chemical
groups, such as stable and unstable detectable isotopes,
radioisotopes, positron-emitting atoms, magnetic resonance labels,
dyes, fluorescent markers, antigenic groups, therapeutic moieties,
or any other moiety that may aid in a prognostic, diagnostic, or
therapeutic application.
For example, in one embodiment, the DAPTZ compound is as defined
herein, but with the additional limitation that the compound
incorporates, is conjugated to, is chelated with, or is otherwise
associated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable
labels, for example, isotopes, radioisotopes, positron-emitting
atoms, magnetic resonance labels, dyes, fluorescent markers,
antigenic groups, or therapeutic moieties.
In one embodiment, the DAPTZ compound is a ligand as well as a
label, e.g., a label for tau protein (or aggregated tau protein),
and incorporates, is conjugated to, is chelated with, or is
otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)
detectable labels.
For example, in one embodiment, the DAPTZ compound is as defined
above, but with the additional limitation that the compound
incorporates, is conjugated to, is chelated with, or is otherwise
associated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable
labels.
Labelled DAPTZ compounds (e.g., when ligated to tau protein or
aggregated tau protein) may be visualised or detected by any
suitable means, and the skilled person will appreciate that any
suitable detection means as is known in the art may be used.
For example, the DAPTZ compound (ligand-label) may be suitably
detected by incorporating a positron-emitting atom (e.g., .sup.11C)
(e.g., as a carbon atom of one or more alkyl group substituents,
e.g., methyl group substituents) and detecting the compound using
positron emission tomography (PET) as is known in the art.
Such .sup.11C labelled DAPTZ compounds may be prepared by adapting
the methods described herein in known ways, for example, in analogy
to the methods described in WO 02/075318 (see FIGS. 11a, 11b, 12)
and WO 2005/030676.
Thus, another aspect of the present invention pertains to a method
of labelling tau protein (or aggregated tau protein) comprising the
step of: (i) contacting the tau protein (or aggregated tau protein)
with a DAPTZ compound that incorporates, is conjugated to, is
chelated with, or is otherwise associated with, one or more (e.g.,
1, 2, 3, 4, etc.) detectable labels.
Another aspect of the present invention pertains to a method of
detecting tau protein (or aggregated tau protein) comprising the
steps of: (i) contacting the tau protein (or aggregated tau
protein) with a DAPTZ compound that incorporates, is conjugated to,
is chelated with, or is otherwise associated with, one or more
(e.g., 1, 2, 3, 4, etc.) detectable labels, and (ii) detecting the
presence and/or amount of said compound bound to tau protein (or
aggregated tau protein).
Another aspect of the present invention pertains to a method of
diagnosis or prognosis of a tau proteinopathy in a subject believed
to suffer from the disease, comprising the steps of: (i)
introducing into the subject a DAPTZ compound capable of labelling
tau protein or aggregated tau protein, particularly tau protein
(e.g., a DAPTZ compound that incorporates, is conjugated to, is
chelated with, or is otherwise associated with, one or more (e.g.,
1, 2, 3, 4, etc.) detectable labels); (ii) determining the presence
and/or amount of said compound bound to tau protein or aggregated
tau protein in the brain of the subject; and (iii) correlating the
result of the determination made in (ii) with the disease state of
the subject.
Another aspect of the present invention pertains to a DAPTZ
compound capable of labelling tau protein or aggregated tau protein
(e.g., a DAPTZ compound that incorporates, is conjugated to, is
chelated with, or is otherwise associated with, one or more (e.g.,
1, 2, 3, 4, etc.) detectable labels), for use in a method of
diagnosis or prognosis of a tau proteinopathy.
Another aspect of the present invention pertains to use of a DAPTZ
compound capable of labelling tau protein or aggregated tau
protein, particularly tau protein (e.g., a DAPTZ compound that
incorporates, is conjugated to, is chelated with, or is otherwise
associated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable
labels), in a method of manufacture of a diagnostic or prognostic
reagent for use in the diagnosis or prognosis of a tau
proteinopathy.
Those skilled in the art will appreciate that instead of
administering DAPTZ ligands/labels directly, they could be
administered in a precursor form, for conversion to the active form
(e.g., ligating form, labelling form) by an activating agent
present in, or administered to, the same subject.
The ligands disclosed herein may be used as part of a method of
diagnosis or prognosis. It may be used to select a patient for
treatment, or to assess the effectiveness of a treatment or a
therapeutic (e.g., an inhibitor of tau protein aggregation)
administered to the subject.
Treatment
The term "treatment," as used herein in the context of treating a
condition, pertains generally to treatment and therapy, whether of
a human or an animal (e.g., in veterinary applications), in which
some desired therapeutic effect is achieved, for example, the
inhibition of the progress of the condition, and includes a
reduction in the rate of progress, a halt in the rate of progress,
regression of the condition, amelioration of the condition, and
cure of the condition. Treatment as a prophylactic measure (i.e.,
prophylaxis, prevention) is also included.
The term "therapeutically-effective amount," as used herein,
pertains to that amount of a DAPTZ compound, or a material,
composition or dosage from comprising a DAPTZ compound, which is
effective for producing some desired therapeutic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
Similarly, the term "prophylactically effective amount," as used
herein, pertains to that amount of a DAPTZ compound, or a material,
composition or dosage from comprising a DAPTZ compound, which is
effective for producing some desired prophylactic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
The term "treatment" includes combination treatments and therapies,
in which two or more treatments or therapies are combined, for
example, sequentially or simultaneously. Examples of treatments and
therapies include, but are not limited to, chemotherapy (the
administration of active agents, including, e.g., drugs, antibodies
(e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic
therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; and gene
therapy.
For example, it may be beneficial to combine treatment with a DAPTZ
compound as described herein with one or more other (e.g., 1, 2, 3,
4) agents or therapies.
The particular combination would be at the discretion of the
physician who would select dosages using his/her common general
knowledge and dosing regimens known to a skilled practitioner.
The agents (i.e., a DAPTZ compound as described here, plus one or
more other agents) may be administered simultaneously or
sequentially, and may be administered in individually varying dose
schedules and via different routes. For example, when administered
sequentially, the agents can be administered at closely spaced
intervals (e.g., over a period of 5-10 minutes) or at longer
intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer
periods apart where required), the precise dosage regimen being
commensurate with the properties of the therapeutic agent(s).
The agents (i.e., a DAPTZ compound as described here, plus one or
more other agents) may be formulated together in a single dosage
form, or alternatively, the individual agents may be formulated
separately and presented together in the form of a kit, optionally
with instructions for their use.
Routes of Administration
The DAPTZ compound, or pharmaceutical composition comprising it,
may be administered to a subject/patient by any convenient route of
administration, whether systemically/peripherally or topically
(i.e., at the site of desired action).
Routes of administration include, but are not limited to, oral
(e.g., by ingestion); buccal; sublingual; transdermal (including,
e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by
a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular
(e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation
therapy using, e.g., via an aerosol, e.g., through the mouth or
nose); rectal (e.g., by suppository or enema); vaginal (e.g., by
pessary); parenteral, for example, by injection, including
subcutaneous, intradermal, intramuscular, intravenous,
intraarterial, intracardiac, intrathecal, intraspinal,
intracapsular, subcapsular, intraorbital, intraperitoneal,
intratracheal, subcuticular, intraarticular, subarachnoid, and
intrasternal (including, e.g., intracatheter injection into the
brain); by implant of a depot or reservoir, for example,
subcutaneously or intramuscularly.
The Subject/Patient
The subject/patient may be an animal, a mammal, a placental mammal,
a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine
(e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a
bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a
horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g.,
a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,
marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,
gibbon), or a human.
Furthermore, the subject/patient may be any of its forms of
development, for example, a foetus.
In one preferred embodiment, the subject/patient is a human.
In one embodiment, the subject/patient is not a human.
Formulations
While it is possible for the DAPTZ compound to be used (e.g.,
administered) alone, it is often preferable to present it as a
composition or formulation.
In one embodiment, the composition is a pharmaceutical composition
(e.g., formulation, preparation, medicament) comprising a DAPTZ
compound, as described herein, and a pharmaceutically acceptable
carrier, diluent, or excipient.
In one embodiment, the composition is a pharmaceutical composition
comprising at least one DAPTZ compound, as described herein,
together with one or more other pharmaceutically acceptable
ingredients well known to those skilled in the art, including, but
not limited to, pharmaceutically acceptable carriers, diluents,
excipients, adjuvants, fillers, buffers, preservatives,
anti-oxidants, lubricants, stabilisers, solubilisers, surfactants
(e.g., wetting agents), masking agents, colouring agents,
flavouring agents, and sweetening agents.
In one embodiment, the composition further comprises other active
agents, for example, other therapeutic or prophylactic agents.
Suitable carriers, diluents, excipients, etc. can be found in
standard pharmaceutical texts. See, for example, Handbook of
Pharmaceutical Additives, 2nd Edition (eds. M. Ash and 1. Ash),
2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA),
Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott,
Williams & Wilkins, 2000; and Handbook of Pharmaceutical
Excipients, 2nd edition, 1994.
Another aspect of the present invention pertains to methods of
making a pharmaceutical composition comprising admixing at least
one [.sup.11C]-radiolabelled DAPTZ compound, as defined herein,
together with one or more other pharmaceutically acceptable
ingredients well known to those skilled in the art, e.g., carriers,
diluents, excipients, etc. If formulated as discrete units (e.g.,
tablets, etc.), each unit contains a predetermined amount (dosage)
of the DAPTZ compound.
The term "pharmaceutically acceptable," as used herein, pertains to
compounds, ingredients, materials, compositions, dosage forms,
etc., which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of the subject in
question (e.g., human) without excessive toxicity, irritation,
allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio. Each carrier, diluent,
excipient, etc. must also be "acceptable" in the sense of being
compatible with the other ingredients of the formulation.
The formulations may be prepared by any methods well known in the
art of pharmacy. Such methods include the step of bringing into
association the DAPTZ compound with a carrier which constitutes one
or more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing into association the
DAPTZ compound with carriers (e.g., liquid carriers, finely divided
solid carrier, etc.), and then shaping the product, if
necessary.
The formulation may be prepared to provide for rapid or slow
release; immediate, delayed, timed, or sustained release; or a
combination thereof.
Formulations suitable for parenteral administration (e.g., by
injection), include aqueous or non-aqueous, isotonic, pyrogen-free,
sterile liquids (e.g., solutions, suspensions), in which the DAPTZ
compound is dissolved, suspended, or otherwise provided (e.g., in a
liposome or other microparticulate). Such liquids may additional
contain other pharmaceutically acceptable ingredients, such as
anti-oxidants, buffers, preservatives, stabilisers, bacteriostats,
suspending agents, thickening agents, and solutes which render the
formulation isotonic with the blood (or other relevant bodily
fluid) of the intended recipient. Examples of excipients include,
for example, water, alcohols, polyols, glycerol, vegetable oils,
and the like. Examples of suitable isotonic carriers for use in
such formulations include Sodium Chloride Injection, Ringer's
Solution, or Lactated Ringer's Injection. Typically, the
concentration of the DAPTZ compound in the liquid is from about 1
ng/ml to about 10 .mu.g/ml, for example from about 10 ng/ml to
about 1 .mu.g/ml. The formulations may be presented in unit-dose or
multi-dose sealed containers, for example, ampoules and vials, and
may be stored in a freeze-dried (lyophilised) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules, and tablets.
Examples of Some Preferred Formulations
One aspect of the present invention pertains to a dosage unit
(e.g., a pharmaceutical tablet or capsule) comprising 20 to 300 mg
of a DAPTZ compound as described herein (e.g., obtained by, or
obtainable by, a method as described herein; having a purity as
described herein; etc.), and a pharmaceutically acceptable carrier,
diluent, or excipient.
In one embodiment, the dosage unit is a tablet.
In one embodiment, the dosage unit is a capsule.
In one embodiment, the amount is 30 to 200 mg.
In one embodiment, the amount is about 30 mg.
In one embodiment, the amount is about 60 mg.
In one embodiment, the amount is about 100 mg.
In one embodiment, the amount is about 150 mg.
In one embodiment, the amount is about 200 mg.
In one embodiment, the pharmaceutically acceptable carrier,
diluent, or excipient is or comprises one or both of a glyceride
(e.g., Gelucire 44/14.RTM.; lauroyl macrogol-32 glycerides PhEur,
USP) and colloidal silicon dioxide (e.g., 2% Aerosil 200.RTM.;
Colliodal Silicon Dioxide PhEur, USP).
Dosage
It will be appreciated by one of skill in the art that appropriate
dosages of the DAPTZ compound, and compositions comprising the
DAPTZ compound, can vary from patient to patient. Determining the
optimal dosage will generally involve the balancing of the level of
therapeutic benefit against any risk or deleterious side effects.
The selected dosage level will depend on a variety of factors
including, but not limited to, the activity of the particular
compound, the route of administration, the time of administration,
the rate of excretion of the compound, the duration of the
treatment, other drugs, compounds, and/or materials used in
combination, the severity of the condition, and the species, sex,
age, weight, condition, general health, and prior medical history
of the patient. The amount of compound and route of administration
will ultimately be at the discretion of the physician,
veterinarian, or clinician, although generally the dosage will be
selected to achieve local concentrations at the site of action
which achieve the desired effect without causing substantial
harmful or deleterious side-effects.
Administration can be effected in one dose, continuously or
intermittently (e.g., in divided doses at appropriate intervals)
throughout the course of treatment. Methods of determining the most
effective means and dosage of administration are well known to
those of skill in the art and will vary with the formulation used
for therapy, the purpose of the therapy, the target cell(s) being
treated, and the subject being treated. Single or multiple
administrations can be carried out with the dose level and pattern
being selected by the treating physician, veterinarian, or
clinician.
In general, a suitable dose of the DAPTZ compound is in the range
of about 100 ng to about 25 mg (more typically about 1 .mu.g to
about 10 mg) per kilogram body weight of the subject per day.
In one embodiment, the DAPTZ compound is administered to a human
patient according to the following dosage regime: about 100 mg, 3
times daily.
In one embodiment, the DAPTZ compound is administered to a human
patient according to the following dosage regime: about 150 mg, 2
times daily.
In one embodiment, the DAPTZ compound is administered to a human
patient according to the following dosage regime: about 200 mg, 2
times daily.
Examples
The following examples are provided solely to illustrate the
present invention and are not intended to limit the scope of the
invention, as described herein.
Chemical Synthesis
Synthesis 1
3-Nitro-10H-phenothiazine
##STR00032##
Sodium nitrite (20.00 g, 210 mmol) was added to a mixture of
10H-phenothiazine (20.00 g, 50 mmol), chloroform (100 cm.sup.3),
and acetic acid (20 cm.sup.3), and the mixture was stirred for 1
hour at room temperature. Acetic acid (20 cm.sup.3) was then added
and the mixture was stirred for a further 18 hours. The suspension
was filtered and washed with acetic acid, ethanol, water, and
finally ethanol to give a purple/brown solid. The residue was
dissolved in hot DMF and allowed to cool before filtering the
di-nitro compound as a purple solid. Concentration of the DMF
solution and washing the precipitate with water and methanol gave
the title mono-nitro compound (15 g, .about.50%) as a brown solid;
v.sub.max (KBr)/cm.sup.-1: 3328 (NH), 3278 (NH), 3229 (NH), 3119
(CH), 3049 (CH), 1557 (NO.sub.2), 1531 (NO.sub.2); .delta..sub.H
(250 MHz; DMSO): 6.64 (5H, m, ArH), 7.68 (1H, d, J 2.5, ArH),
7.79-7.84 (1H, dd, J 2.75, 6.5, ArH; .delta..sub.C (62.9 MHz;
DMSO): 113.3 (ArC), 115.3 (ArC), 116.9 (ArC), 121.8 (ArC), 123.6
(ArC), 123.7 (ArC), 124.6 (ArC), 126.4 (ArC), 128.1 (ArC), 138.8
(ArC), 141.0 (ArC), 147.8 (ArC).
Synthesis 2
3,11-Dinitro-10H-phenothiazine
##STR00033##
The procedure for the synthesis of 3-nitro-10H-phenothiazine was
followed using 3-nitro-10H-phenothiazine (10.00 g, 41 mmol),
chloroform (40 cm.sup.3), acetic acid (2.times.10 cm.sup.3), and
sodium nitrite (11.86 g, 173 mmol). The residue obtained was
recrystallised from DMF to yield the title di-nitro compound (6.60
g 56%) as purple needles; v.sub.max (KBr)/cm.sup.-1: 3331 (NH),
3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH), 1602 (NO.sub.2), 1558
(NO.sub.2); .delta..sub.H (250 MHz; DMSO): 6.73-6.76 (2H, d, J 9,
ArH), 7.78 (2H, s, ArH, 7.89-7.85 (2H, d, J 9, ArH).
Synthesis 3
1-(3,7-Dinitro-phenothiazin-10-yl)-ethanone
##STR00034##
A solution of 3,11-dinitro-10H-phenothiazine (3.00 g, 10.37 mmol),
acetic anhydride (15.88 g, 155.50 mmol), and pyridine (30 cm.sup.3)
was stirred at reflux for 18 hours. The warm solution was then
carefully poured over ice water. A precipitate formed and was
filtered, dissolved in dichloromethane, dried over magnesium
sulphate, filtered, and concentrated to give a brown/orange solid,
which was purified by column chromatography (SiO.sub.2, ethyl
acetate:petroleum ether, 2:3, loaded as a dichloromethane solution)
to give the title compound (2.46 g, 71%) as a light yellow solid
which can be recrystallised from acetone to give light yellow
needles; v.sub.max (KBr)/cm.sup.-1: 3091 (CH), 3063 (CH), 1680
(C.dbd.O), 1575 (NO.sub.2), 1510 (NO.sub.2); .delta..sub.H (250
MHz; CDCl.sub.3): 2.28 (3H, s, CH.sub.3), 7.65-7.69 (2H, d, J 9,
ArH), 8.22-8.26 (2H, dd, J 2.75, 8.75, ArH), 8.33-8.32 (2H, d, J
2.5, ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3): 168.2 (C.dbd.O),
146.3 (ArC), 143.3 (ArC), 133.6 (ArC), 127.8 (ArC), 123.4 (ArC),
122.9 (ArC), 23.1 (CH.sub.3); m/z (ES) 331.0 (80%, [M].sup.+).
Synthesis 4
1-(3,7-Diamino-phenothiazin-10-yl)-ethanone
##STR00035##
A mixture of 1-(3,7-dinitro-phenothiazin-10-yl)-ethanone (2 g, 6.04
mmol), tin (II) chloride dihydrate (14.17 g, 62.8 mmol), and
ethanol (50 cm.sup.3) was heated to reflux and stirred at this
temperature for 5 hours. The mixture was then cooled to room
temperature and poured over ice water. The pH was adjusted to 7
with 5% sodium hydrogen carbonate before the product was extracted
with ethyl acetate (3.times.50 cm.sup.3). The extracts were washed
with brine and dried over magnesium sulphate, filtered, and
concentrated to give the title compound (1.64 g, 100%) as a purple
blue solid; v.sub.max(KBr)/cm.sup.-1: 3445 (NH), 3424 (NH), 3368
(NH), 3322 (NH), 3203 (NH), 3054 (CH), 2995 (CH), 1706 (C.dbd.O),
1650 (NO.sub.2), 1590 (NO.sub.2); .delta..sub.H (250 MHz;
CDCl.sub.3): 2.01 (3H, s, CH.sub.3), 5.09-5.43 (4H, brd s, NH),
6.47-6.51 (2H, dd, J 1.5, 8.25, ArH), 6.61 (2H, s, ArH), 7.11-7.15
(2H, d, J 8, ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3): 169.1
(C.dbd.O), 147.2 (ArC), 128.1 (ArC), 127.6 (ArC), 127.3 (ArC),
112.3 (ArC), 111.5 (ArC), 22.6 (CH.sub.3); m/z (ES) 293.9 (95%,
[M+H, Na].sup.+), 272.0 (20%, [M+H].sup.+), 227.9 (100%, [M+H,
--Ac].sup.+).
Synthesis 5
3,7-Diamino-phenothiazine bis(hydrogen chloride) (B4)
##STR00036##
1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g, 0.921 mmol)
was dissolved in aqueous hydrochloric acid (5 N, 10 cm.sup.3) and
the solution was heated to reflux and stirred for 30 minutes.
Concentration of the reaction mixture gave the title compound as a
light blue solid. .delta..sub.H (250 MHz; D.sub.2O): 6.60 (2H, brd
d, ArH), 7.07 (4H, brd s, ArH).
Synthesis 6
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00037##
1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g 0.92 mmol) was
dissolved in DMSO (3 cm.sup.3). Toluene (10 cm.sup.3), iodomethane
(1.96 g, 13.8 mmol), tetrabutylammoniun bromide (50 mg), and
finally aqueous sodium hydroxide solution (50%, 1.25 cm.sup.3) were
added. The mixture was stirred at room temperature for 2 hours.
Additional aqueous sodium hydroxide (50%, 1.25 cm.sup.3) and
iodomethane (1.96 g, 13.8 mmol) were then added. The mixture was
allowed to stir for a further 3 hours at room temperature before a
third aliquot of aqueous sodium hydroxide (50%, 1.25 cm.sup.3) and
iodomethane (1.96 g, 13.8 mmol) were added and the mixture stirred
for a further 18 hours. The thick suspension was washed with water
(3.times.75 cm.sup.3) and the toluene extract collected. The water
was extracted with dichloromethane (3.times.50 cm.sup.3) and the
extracts combined with the toluene, and dried over magnesium
sulphate, filtered, and concentrated to give a deep purple solid.
The residue was purified by column chromatography (SiO.sub.2, ethyl
acetate:petroleum ether, 2:3, loaded as a dichloromethane solution)
to give the title compound product (0.12 g, 40%) as a light purple
solid; v.sub.max(KBr)/cm.sup.-1: 2910 (CH), 2876 (CH), 2856 (CH),
2799 (CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3): 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3): 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3).
Synthesis 7
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride) (B3)
##STR00038##
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.84
mmol) was dissolved in aqueous hydrochloric acid (5 N, 15
cm.sup.3), and the solution was heated to reflux temperature and
stirred for 30 minutes. Concentration of the reaction mixture gave
the title compound as a green/blue solid; .delta..sub.H (250 MHz;
D.sub.2O): 3.18 (12H, s, NCH.sub.3), 6.67 (2H, d, J8.5, ArH), 7.16
(4H, brd s, ArH); .delta..sub.C (62.9 MHz; D.sub.2O): 144.3 (ArC),
138.9 (ArC), 122.4 (ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC),
48.9 (NCH.sub.3).
Synthesis 8
Methylthioninium Iodide
##STR00039##
To a round bottom flask was added methylthioninium chloride (MTC,
Methylene Blue) (2 g, 6.25 mmol) and water (50 cm.sup.3) and the
mixture stirred for 10 minutes or until the solid dissolved.
Potassium iodide (1.56 g, 9.4 mmol) was then added to the mixture
and a green black suspension formed. The reaction was heated to
boiling and allowed to cool naturally giving the title compound
(2.03 g, 79%) as bright green needles. Anal. Calcd for
C.sub.16H.sub.18N.sub.3SI: C, 46.72; H, 4.41; N, 10.22; S, 7.80; I,
30.85. Found: C, 46.30; H, 4.21; N, 10.14; S, 7.86; I, 29.34.
Synthesis 9
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
iodide) (B6)
##STR00040##
To a round bottom flask was added methylthioninium iodide (2 g,
4.86 mmol), ethanol (100 cm.sup.3) and ethyl iodide (75.8 g, 486
mmol) and the mixture was heated at reflux for 18 hours where the
colour changed from green/blue to brown with a yellow precipitate.
Once cooled to room temperature, the mixture was filtered and
washed with diethylether (20 cm.sup.3) to give the title compound
(1.99 g, 76%) as a light green solid. .delta..sub.H (250 MHz;
D.sub.2O): 3.20 (12H, s, NCH.sub.3), 6.76 (2H, d, J 8.5, ArH), 7.22
(2H, brd s, ArH); .delta..sub.C (62.9 MHz; D.sub.2O): 145.0 (ArC),
139.3 (ArC), 122.6 (ArC), 121.1 (ArC), 120.9 (ArC), 117.9 (ArC),
48.9 (NCH.sub.3).
Synthesis 10
1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone
##STR00041##
To a dry 25 cm.sup.3 round bottom flask was added ethylthioninium
zinc chloride (0.5 g, 1.13 mmol) and ethanol (10 cm.sup.3).
Phenylhydrazine (0.134 g, 1.24 mmol) was then added dropwise under
an atmosphere of nitrogen. The mixture was stirred 25.degree. C.
for 1 hour and concentrated under high vacuum. Pyridine (50
cm.sup.3) and acetic anhydride was added and the mixture stirred
for 18 hours at 60.degree. C. The solution was opened to ice/water
(250 cm.sup.3) and the organics were extracted into ethyl acetate
(3.times.50 cm.sup.3). The extracts were washed with saturated
copper sulphate solution and dried over magnesium sulphate,
filtered, and concentrated to give the crude product as a brown
oil, which was purified using flash column chromatography with an
eluent of 40% ethylacetate:60% petroleum spirit 40-60.degree. C.
and silica 40-63.mu. 60 .ANG. to give the title compound (0.18 g,
41%) as a green glassy solid. .delta..sub.H (250 MHz; CDCl.sub.3):
7.0-7.5 (2H, brds, ArH), 6.64 (2H, s, ArH), 6.52 (2H, d, ArH), 3.35
(8H, q, 7, NCH.sub.2), 2.18 (3H, s, CH.sub.3), 1.16 (12H, t, 7,
CH.sub.3); .delta..sub.C (62.9 MHz; CDCl.sub.3): 12.5 (CH.sub.3),
22.9 (CH.sub.3), 44.6 (NCH.sub.2), 110.1 (ArC), 127.4 (ArC), 146.5
(ArC), 170.2 (C.dbd.O).
Synthesis 11
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00042##
To a 25 cm.sup.3 round bottom flask was added
3,7-diethylamino-10-acetyl-phenothiazine (0.125 g, 0.33 mmol) and
aqueous hydrochloric acid (5 M, 5 cm.sup.3). The mixture was heated
at 100.degree. C. for 2 hours before cooling to room temperature
and was concentrated to give the title compound (0.11 g, 81%) as a
yellow green glassy solid. .delta..sub.H (250 MHz; CD.sub.3OD):
7.07 (4H, brd, ArH), 6.65 (2H, brd, ArH), 3.35 (8H, brd,
NCH.sub.2), 0.97 (12H, brd, CH.sub.3); .delta..sub.C (62.9 MHz;
CD.sub.3OD): 10.8 (CH.sub.3), 55.1 (NCH.sub.2), 116.6 (ArC), 120.4
(ArC), 121.5 (ArC), 123.6 (ArC), 132.6 (ArC), 144.5 (ArC).
Synthesis 12
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00043##
Synthesis using methylhydrazine/pyridine in two pots. To a 250
cm.sup.3 round bottom flask placed under an atmosphere of argon was
added methylthioninium chloride trihydrate (26.74 mmol, 10 g),
ethanol (100 cm.sup.3) and methylhydrazine (58.83 mmol, 2.71 g).
The mixture was heated to 40.degree. C. and stirred for 2 hours.
The yellow/green suspension was cooled to 5.degree. C. and filtered
under argon, washed with ethanol (20 cm.sup.3) and dried to give
leuco-methylene blue as a light green solid. To the leuco product
was added acetic anhydride (40 cm.sup.3) and pyridine (10 cm.sup.3)
and the solution was heated at 100.degree. C. for 18 hours. The
cooled mixture was then poured carefully over ice water while
stirring to give a precipitate, which was filtered, washed with
water, and dried at 60.degree. C. for 2 hours to yield the title
compound (5.82 g, 66%) as a light brown solid. Mp 137.degree. C.;
v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 13
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00044##
Synthesis using methylhydrazine/Hunig's base in one pot. To a 5000
cm.sup.3 reactor vessel under an atmosphere of nitrogen was added
methylthioninium chloride trihydrate (0.54 mol, 200 g) and
acetonitrile (1000 cm.sup.3). Methylhydrazine (1.07 mol, 49.36 g)
was added dropwise at 1.5 mL per minute. The temperature of the
mixture increased to 32.degree. C. and was stirred for 20 minutes.
The yellow/green suspension had acetic anhydride (5.35 mol, 541 g)
added and then Hunig's base (diisopropylethylamine) (1.55 mol, 200
g) was added. The mixture was heated at 90.degree. C. for 2 hours.
The cooled mixture was then poured carefully into ice water (2000
cm.sup.3) in ten 200 cm.sup.3 portions while stirring to give a
precipitate. The precipitate was stirred for 45 minutes before it
was filtered, washed with water (3.times.250 cm.sup.3), and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (2750 cm.sup.3) to yield the title compound (112.1 g, 64%)
as a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 14
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00045##
Synthesis using methylhydrazine/pyridine in one pot. To a 250
cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (26.74 mmol, 10 g) and
acetonitrile (50 cm.sup.3). Methylhydrazine (53.5 mmol, 2.46 g) was
added in four equal portions over a 30 minutes time period. The
temperature of the mixture was maintained at 35.degree. C. with a
cold water bath and was stirred for 30 minutes. The yellow/green
suspension had acetic anhydride (267 mmol, 27.3 g) and pyridine
(80.2 mmol, 6.35 g) was added. The mixture was heated at 90.degree.
C. for 2 hours. The cooled mixture was then poured carefully into
ice water (200 cm.sup.3) in ten equal portions while stirring to
give a precipitate. The precipitate was stirred for 30 minutes
before it was filtered, washed with water (3.times.50 cm.sup.3) and
air dried for 30 minutes. The crude material was crystallised from
hot ethanol (120 cm.sup.3) to yield the title compound (5.97 g,
68%) as a light grey solid. Mp 137.degree. C.;
v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 15
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00046##
Synthesis using sodium borohydride/pyridine in one pot. To a 500
cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (0.134 mol, 50 g) and
acetonitrile (250 cm.sup.3). Sodium borohydride (0.174 mol, 6.6 g)
was added in four equal portions over a 30 minute time period. The
temperature of the mixture was maintained at 35.degree. C. with a
cold water bath and was stirred for 30 minutes. The yellow/green
suspension had acetic anhydride (0.535 mol, 55 g) and pyridine
(0.174 mol, 13.76 g) added. The mixture was heated at 90.degree. C.
for 2 hours. The cooled mixture was then poured carefully into ice
water (250 cm.sup.3) in ten equal portions while stirring to give a
precipitate. The precipitate was stirred for 30 minutes before it
was filtered, washed with water (3.times.50 cm.sup.3), and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (500 cm.sup.3) to yield the title compound (26.7 g, 61%) as
a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.c (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 16
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00047##
Synthesis using sodium borohydride/Hunig's base in one pot. To a
500 cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (80.2 mmol, 30 g) and
acetonitrile (150 cm.sup.3). Sodium borohydride (104 mmol, 3.94 g)
was added in four equal portions over a 30 minute time period. The
temperature of the mixture was maintained at 35.degree. C. with a
cold water bath and was stirred for 30 minutes. The yellow/green
suspension had acetic anhydride (321 mmol, 32.75 g) and Hunig's
base (diisopropylethylamine) (120 mmol, 15.55 g) added. The mixture
was heated at 90.degree. C. for 2 hours. The cooled mixture was
then poured carefully into ice water (200 cm.sup.3) in ten equal
portions while stirring to give a precipitate. The precipitate was
stirred for 30 minutes before it was filtered, washed with water
(3.times.50 cm.sup.3), and air dried for 30 minutes. The crude
material was crystallised from hot ethanol (300 cm.sup.3) to yield
the title compound (13.55 g, 52%) as a light grey solid. Mp
137.degree. C.; v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856
(CH), 2799 (CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 17
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00048##
Synthesis using hydrazine monohydrate/pyridine in one pot. To a 250
cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (26.74 mmol, 10 g) and
acetonitrile (50 cm.sup.3). Hydrazine monohydrate (58.8 mmol, 2.95
g) was added and the mixture was heated to reflux and stirred for
10 minutes before cooling to 25.degree. C. The yellow/green
suspension had acetic anhydride (424 mmol, 43.3 g) and pyridine
(124 mmol, 9.78 g) added. The mixture was heated at 90.degree. C.
for 2 hours. The cooled mixture was then poured carefully into ice
water (100 cm.sup.3) in ten equal portions while stirring to give a
precipitate. The precipitate was stirred for 30 minutes before it
was filtered, washed with water (3.times.50 cm.sup.3), and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (100 cm.sup.3) to yield the title compound (4.87 g, 56%) as
a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 18
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00049##
Synthesis using hydrazine monohydrate/Hunig's base in one pot. To a
250 cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (80.2 mmol, 30 g) and
acetonitrile (150 cm.sup.3). Hydrazine monohydrate (176.5 mmol,
8.84 g) was added and the mixture was heated to reflux and stirred
for 10 minutes before cooling to 25.degree. C. The yellow/green
suspension had acetic anhydride (794 mmol, 81.2 g) and Hunig's base
(diisopropylethylamine) (232 mmol, 29.97 g) added. The mixture was
heated at 90.degree. C. for 2 hours. The cooled mixture was then
poured carefully into ice water (400 cm.sup.3) in ten equal
portions while stirring to give a precipitate. The precipitate was
stirred for 30 minutes before it was filtered, washed with water
(3.times.100 cm.sup.3), and air dried for 30 minutes. The crude
material was crystallised from hot ethanol (400 cm.sup.3) to yield
the title compound (17.15 g, 65%) as a light grey solid. Mp
137.degree. C.; v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856
(CH), 2799 (CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 19
3,11-Dinitro-10H-phenothiazine
##STR00050##
10H-Phenothiazine (20.00 g, 100 mmol), dichloromethane (100
cm.sup.3) and acetic acid (40 cm.sup.3) had sodium nitrite (20.07
g, 300 mmol) added and the mixture was stirred for 10 minutes at
room temperature. Additional acetic acid (40 cm.sup.3),
dichloromethane (100 cm.sup.3) and sodium nitrite (20.07 g, 300
mmol) were then added. A further 120 cm.sup.3 of acetic acid was
added to try and break up the thick reaction mixture. The mixture
was stirred for 3 hours. The suspension was filtered and washed
with 100 cm.sup.3 each of ethanol, water, and finally ethanol to
give a purple/brown solid. The residue was stirred in hot DMF and
allowed to cool before filtering the dinitro product, which was
washed with ethanol (150 cm.sup.3) and dried to give the title
compound (24.88 g, 86%) as a brown solid; v.sub.max(KBr)/cm.sup.-1
3331 (NH), 3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH), 1602
(NO.sub.2), 1558 (NO.sub.2); .delta..sub.H (250 MHz; DMSO)
6.73-6.76 (2H, d, J 9, ArH), 7.78 (2H, s, Arm), 7.89-7.85 (2H, d, J
9, ArH).
Synthesis 20
1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone
##STR00051##
To a 250 cm.sup.3 round bottom flask under an atmosphere of
nitrogen was added ethylthioninium nitrate monohydrate (7.13 mmol,
3 g) and acetonitrile (20 cm.sup.3). Hydrazine monohydrate (16.4
mmol, 0.82 g) was added and the mixture was heated to reflux and
stirred for 10 minutes before cooling to 25.degree. C. The brown
solution had acetic anhydride (114 mmol, 11.65 g) and Hunig's base
(diisopropylethylamine) (21.4 mmol, 2.77 g) was added. The mixture
was heated at 90.degree. C. for 2 hours. The cooled mixture was
then poured carefully into ice water (40 cm.sup.3) in ten equal
portions while stirring to give a precipitate. The precipitate was
stirred for 30 minutes before it was filtered, washed with water
(3.times.25 cm.sup.3) and air dried for 30 minutes. The crude
material was crystallised from hot ethanol (50 cm.sup.3) to yield
the title compound (1.73 g, 63%) as a light grey solid.
.delta..sub.H (250 MHz; CDCl.sub.3) 7.0-7.5 (2H, brds, ArH), 6.64
(2H, s, ArH), 6.52 (2H, d, ArH), 3.35 (8H, q, 7, NCH.sub.2), 2.18
(3H, s, CH.sub.3), 1.16 (12H, t, 7, CH.sub.3); .delta..sub.C (62.9
MHz; CDCl.sub.3) 12.5 (CH.sub.3), 22.9 (CH.sub.3), 44.6
(NCH.sub.2), 110.1 (ArC), 127.4 (ArC), 146.5 (ArC), 170.2
(C.dbd.O).
Synthesis 21
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00052##
To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30
mmol), ethanol (5 cm.sup.3), and hydrochloric acid (37%, 1.3
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, the mixture was concentrated
giving the title compound (0.54 g, 100%) as a light green glass.
.delta..sub.H (250 MHz; CD.sub.3OD) 7.07 (4H, brd, ArH), 6.65 (2H,
brd, ArH), 3.35 (8H, brd, NCH.sub.2), 0.97 (12H, brd, CH.sub.3);
.delta..sub.C (62.9 MHz; CD.sub.3OD) 10.8 (CH.sub.3), 55.1
(NCH.sub.2), 116.6 (ArC), 120.4 (ArC), 121.5 (ArC), 123.6 (ArC),
132.6 (ArC), 144.5 (ArC).
Synthesis 22
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00053##
To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30
mmol), ethanol (5 cm.sup.3), and hydrobromic acid (48%, 0.75
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, the mixture was concentrated
giving the title compound (0.65 g, 100%) as a light yellow glass.
.delta..sub.H (250 MHz; D.sub.2O) 7.05 (4H, brd, ArH), 6.79 (2H,
brd d, ArH), 3.43 (8H, brd, NCH.sub.2), 1.05 (12H, brd t,
CH.sub.3); .delta..sub.C (62.9 MHz; D.sub.2O) 12.3 (CH.sub.3), 56.2
(NCH.sub.2), 117.9 (ArC), 121.4 (ArC), 122.4 (ArC), 124.5 (ArC),
133.5 (ArC), 145.1 (ArC).
Synthesis 23
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00054##
To a round bottom flask was added
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05
mmol), ethanol (10 cm.sup.3), and hydrochloric acid (37%, 3
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, diethyl ether was added while
stirring until a constant turbid solution was obtained. After some
time, a precipitate formed, which was filtered and washed with
diethyl ether (10 cm.sup.3) giving the title compound (0.98 g, 90%)
as a light green solid. Mp (dec) 230.degree. C.;
v.sub.max(KBr)/cm.sup.-1 3500-3229 (NH), 3061 (CH), 3021 (CH), 2948
(CH), 2879 (CH), 2679 (CH), 2601 (CH), 1604 (CH), 1483 (CH), 1318
(CH); .delta..sub.H (250 MHz; D.sub.2O) 3.18 (12H, s, NCH.sub.3),
6.67 (2H, d, J 8.5, ArH), 7.16 (4H, brd s, ArH; .delta..sub.C (62.9
MHz; D.sub.2O) 144.3 (ArC), 138.9 (ArC), 122.4 (ArC), 120.8 (ArC),
120.7 (ArC), 117.6 (ArC), 48.9 (NCH.sub.3); m/z (ES) 286.1 (100%,
[M-H, 2Cl].sup.+), 285.1 (40%), 284.1 (41%, [M-3H, 2Cl].sup.+).
Synthesis 24
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00055##
To a round bottom flask was added
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05
mmol), ethanol (10 cm.sup.3), and hydrobromic acid (48%, 4
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, a precipitate formed, which was
filtered and washed with diethyl ether (10 cm.sup.3) giving the
product (1.22 g, 89%) as a light mustard solid. Mp (dec)
230.degree. C.; v.sub.max(KBr)/cm.sup.-1 3500-3229 (NH), 3061 (CH),
3021 (CH), 2948 (CH), 2879 (CH), 2679 (CH), 2601 (CH), 1604 (CH),
1483 (CH), 1318 (CH); .delta..sub.H (250 MHz; D.sub.2O) 3.18 (12H,
s, NCH.sub.3), 6.66 (2H, d, J 8.75, ArH), 7.15 (4H, s, ArH);
.delta..sub.C (62.9 MHz; D.sub.2O) 144.3 (ArC), 138.9 (ArC), 122.4
(ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC), 48.9 (NCH.sub.3).
Synthesis 25
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00056##
To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (1.0 g, 2.60
mmol), methanol (10 cm.sup.3), and hydrobromic acid (48%, 2.94
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to 5.degree. C., the mixture had diethyl ether added,
giving a cloudy solution. The solution was stirred for 30 minutes
and gave the title compound (0.83 g, 63%) as a light yellow solid.
.delta..sub.H (250 MHz; D.sub.2O) 7.05 (4H, brd, ArH), 6.79 (2H,
brd d, ArH), 3.43 (8H, brd, NCH.sub.2), 1.05 (12H, brd t,
CH.sub.3); .delta..sub.C (62.9 MHz; D.sub.2O) 12.3 (CH.sub.3), 56.2
(NCH.sub.2), 117.9 (ArC), 121.4 (ArC), 122.4 (ArC), 124.5 (ArC),
133.5 (ArC), 145.1 (ArC).
Stability Studies
The DAPTZ compounds of the present invention are stably reduced
(i.e., are in a stably reduced form). For example, they are stable
in the solid form, for example, for at least 1 week, e.g., at least
2 weeks, e.g., at least 1 month, e.g., at least 2 months, e.g., at
least 1 year (e.g., at room temperature, e.g., 18-25.degree. C.,
e.g., in a sealed container).
One sample of compound B3
(N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)), in solid form, was found to be substantially reduced
even after 2 years in storage.
Once the DAPTZ compounds are dissolved in water (i.e., in the form
of an aqueous solution), they slowly oxidize (giving the solution a
blue colour), typically over a period of 1 to 3 hours.
The stability of two DAPTZ compounds of the present invention was
studied, specifically, B3
(N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)), and B6
(N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
iodide)). MTC was used as a standard.
The compounds were weighed into universal containers. Enough water
was added to yield a 1 mM solution, and the mixture stirred to
dissolve the solid. The absorbance was determined at 610 nm and 665
nm for 50 .mu.L samples (in triplicate) of each of the solutions at
various time points. The initial time point taken was at 10 minutes
as the compounds took time to dissolve completely. A UV/visible
spectrum was also recorded at time points 20 minutes, 3 hours, and
18 hours.
The percent reduced form (%) was calculated assuming that the 10
minute reading for MTC represented 0% reduced, and that a blank
represented 100% reduced (colourless).
FIG. 1 is a graph of the percent reduced form (%) versus time
(minutes) for each of three compounds, B1, B3, and B6, as
determined using absorbance at 665 nm.
FIG. 2 is a graph of the percent reduced form (%) versus time
(minutes) for each of three compounds, B1, B3, and B6, as
determined using absorbance at 610 nm.
FIGS. 3A, 3B, and 3C show the UV/visible absorption spectra for
aqueous samples of each of three compounds, B1 (open circles), B3
(open squares), and B6 (open triangles), after 20 minutes (FIG.
3A), 3 hours (FIG. 3B), and 18 hours (FIG. 3C).
These data demonstrate that the DAPTZ compounds (stabilized reduced
forms) remain substantially stable (>50%) for at least 1 hour,
and that compound B6 remains substantially stable (>50%) for
almost 3 hours. However, after about 18 hours, the compounds are
not significantly different from MTC. See, for example, FIG. 3C,
where the spectra are almost indistinguishable.
Additionally, the rate of autoxidation was found to be slower for
the "iodide" compound (Compound B6) as compared to the "chloride"
compound (Compound B3), suggesting that the rate of autoxidation
depends upon the counterion. Although the difference in rate was
small, it may be significant in drug formulation. Other salts may
be more stable against oxidation.
A batch of N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine
bis(hydrogen bromide) was prepared in April 2006 and analysed by
NMR. After 10 months of storage in the dark at room temperature,
the solid material was analysed once more and the NMR data was
found to be identical. The colour of the solid remained consistent
over time. It appears that the molecule, in this form, is stable
under these conditions over this time period.
Biological Studies
Methods: In vitro Assay for Establishing B50
These methods are described in detail in WO 96/30766. Briefly, a
fragment of tau corresponding to the core repeat domain, which has
been adsorbed to a solid phase substrate, is able to capture
soluble full-length tau and bind tau with high affinity. This
association confers stability against proteolytic digestion of the
aggregated tau molecules. The process is self-propagating, and can
be blocked selectively by prototype pharmaceutical agents.
More specifically, truncated tau (residues 297-390; dGA) diluted in
carbonate buffer (pH 9.6) was bound to the assay plate, and
full-length tau (T40) was added in the aqueous phase. The aqueous
phase binding buffer contained 0.05% Tween-20 and 1% gelatine in
phosphate-buffered saline (pH 7.4). Bound tau was detected using
mAb 499 that recognises an N-terminal epitope within the aqueous
phase full-length tau but that fails to recognise the solid
phase-bound truncated tau fragment.
The concentration of compound required to inhibit the tau-tau
binding by 50% is referred to as the B50 value.
Methods: Cell-Based Assay for Establishing EC50
These methods are described in more detail in WO 02/055720.
Briefly, fibroblast cells (3T6) express full-length tau ("T40")
under control of an inducible promoter, and low constitutive levels
of the PHF-core tau fragment (12 kD fragment). When T40 expression
is induced, it undergoes aggregation-dependent truncation within
the cell, N-terminally at .about..alpha..alpha. 295 and
C-terminally at .about..alpha..alpha. 390, thereby producing higher
levels of the 12 kD PHF-core domain fragment. Production of the 12
kD fragment can be blocked in a dose-dependent manner by
tau-aggregation inhibitors. Indeed, the quantitation of inhibitory
activity of compounds with respect to proteolytic generation of the
12 kD fragment within cells can be described entirely in terms of
the same parameters that describe inhibition of tau-tau binding in
vitro. That is, the extent of proteolytic generation of the 12 kD
fragment within cells is determined entirely by the extent of
tau-tau binding through the repeat domain. The availability of the
relevant proteases within the cell is non-limiting.
Results are expressed as the concentration at which there is a 50%
inhibition of generation of the 12 kD fragment. This is referred to
as the EC50 value.
Methods: Toxicity in Cells (LD50) and Therapeutic Index (RxI)
Toxicity of the compounds described herein was assessed in the cell
based assay used to assess EC50. Toxicity was measured by cell
numbers determined after 24 hours exposure to the compound using a
lactate dehydrogenase assay kit TOX-7 (Sigma Biosciences) according
to the manufacturer's instructions after lysis of remaining cells.
Alternatively, a kit from Promega UK (CytoTox 96) was used, again
according to the manufacturer's instructions.
The therapeutic index (RxI) was calculated as: RxI=LD50/EC50.
The data are summarised in the following Table.
TABLE-US-00002 Biological Data Compound B50 EC50 LD50 RxI B3 57.3
0.50 44.0 88 B4 23.5 2.23 -- -- B6 494 0.38 115 303 MTC 218 0.59
65.0 110 B3: N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine
bis(hydrogen chloride). B4: 10H-phenothiazine-3,7-diamine
bis(hydrogen chloride). B6:
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
iodide). MTC: Methylthioninium chloride.
Partition Coefficient Studies
It is well known that the partition coefficient for a drug in an
organic phase/water system (typically an n-octanol/water system),
usually reported as the logarithm (i.e., log.sub.10P), is a good
indicator for the biological activity of that drug. See, e.g.,
Hansch, C., et al., 1964, J. Am. Chem. Soc., Vol. 86, pp.
1616-1626; Kubinyi, H., 1977, J. Med. Chem., Vol. 20, pp. 625-629.
It is believed that this is because the absorption of a compound
depends on its partition between the biological membrane and the
aqueous phase. Partition coefficients are also useful in separation
techniques and in the prediction of the solubility of drugs.
In the context of drug-like substances, hydrophobicity is related
to absorption, bioavailability, hydrophobic drug-receptor
interactions, metabolism, and toxicity. Low hydrophilicities, and
therefore high log.sub.10P values, may cause poor absorption or
permeation. It has been shown for compounds to have a reasonable
probability of being well absorbed, their log.sub.10P value must
not be greater than 5.0. The distribution of calculated log.sub.10P
values of more than 3000 drugs on the market underlines this
fact.
Many methods for determining partition coefficients are known. In
this study, aqueous solutions of selected compounds were shaken
with n-octanol and aliquots concentrations in each phase determined
by visible spectrophotometry. The log.sub.10P was then calculated
for each compound, using the following formula:
log.sub.10P=log.sub.10[Drug].sub.octanol-log.sub.10[Drug].sub.water=log.s-
ub.10([Drug].sub.octanol/[Drug].sub.water)
The data are summarised in the following table. The DAPTZ compounds
of the present invention were found to have log.sub.10P values
expected for drug-like molecules.
TABLE-US-00003 Partition Coefficient Data .lamda..sub.max
Absorbance for Absorbance for Compound (octanol/water) n-octanol
phase water phase Log.sub.10P MTC 665/660 0.217 4.83 -1.35 B3
664/660 0.083 0.111 -0.13 B6 658.4/662.4 0.179 0.563 -0.498 B3:
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride). B6: N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine
bis(hydrogen iodide). MTC: Methylthioninium chloride.
Crystal Structure
FIG. 4 shows the crystal structure of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide). The crystal structure shows three crystallographically
distinct bromide ions. Br1 and Br2 occupy special positions with
2-fold symmetry, whereas the organic main molecule and Br3 occupy
general positions. Hence, the overall stoichiometry is
C.sub.16H.sub.21N.sub.3SBr.sub.2.
FIG. 5 shows the side-on view of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide) and reveals the non-planarity; the dihedral angle between
the outer benzene rings is 11.0 (3) degrees.
FIG. 6 shows part of one helical column of
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide) molecules in the crystal.
* * * * *